Histology and ultrastructure of the salivary glands in Bulla striata (Mollusca, Opisthobranchia)

Abstract. The ribbon‐shaped salivary glands in Bulla striata were studied with light microscopy and transmission electron microscopy (TEM). Secretion is produced in tubules formed by two types of secretory cells, namely granular mucocytes and vacuolated cells, intercalated with ciliated cells. A central longitudinal duct lined by the same cell types collects the secretion and conducts it to the buccal cavity. In granular mucocytes, the nucleus is usually central and the secretory vesicles contain oval‐shaped granular masses attached to the vesicle membrane. Glycogen granules can be very abundant, filling the space around the secretory vesicles. These cells are strongly stained by PAS reaction for polysaccharides. Their secretory vesicles are also stained by Alcian blue, revealing acidic mucopolysaccharides, and the tetrazonium reaction detects proteins in minute spots at the edge of the vesicles, corresponding to the granular masses observed in TEM. Colloidal iron staining for acidic mucopolysaccharides in TEM reveals iron particles in the electron‐lucent region of the vesicles, while the granular masses are free of particles. In vacuolated cells, which are thinner and less abundant than the granular mucocytes, the nucleus is basal and the cytoplasm contains large electron‐lucent vesicles. These vesicles are very weakly colored by light microscopy techniques, but colloidal iron particles could be observed within them. The golf tee‐shaped ciliated cells contain some electron‐dense lysosomes in the apical region. In these cells, the elongated nucleus is subapically located, and bundles of microfibrils are common in the slender cytoplasmic stalk that reaches the basal lamina. The morphological, histochemical, and cytochemical data showed some similarities between salivary glands in B. striata and Aplysia depilans. These similarities could reflect the phylogenetic relationship between cephalaspidean and anaspidean opisthobranchs or result from a convergent adaptation to an identical herbivorous diet.     

The roles of apoptosis and mitosis in atrophy of the rat sublingual gland

The roles of apoptosis and mitosis of acinar and duct cells in the atrophy of the sublingual gland of rat induced by double duct ligation was investigated using immunohistochemistry for proliferating cell nuclear antigen (PCNA), terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end labeling (TUNEL), and transmission electron microscopy (TEM). Many PCNA-positive duct cells were observed 3 days after duct ligation, and the numbers decreased thereafter. At 3 and 5 days, several TUNEL-positive acinar cells were observed and typical apoptotic acinar cells were identified by TEM. Necrotic acinar cells were also observed ultrastructurally. After 7 days, there were few acini but many ducts, as well as many structures representing transition from acinus to duct. These observations demonstrate that acinar cell loss by apoptosis and duct cell proliferation by mitosis occur in atrophic sublingual glands as well as in other atrophic salivary glands. In addition, it appears that the transition from acinar to duct cell and the necrosis of acinar cells play important roles in the atrophy of the sublingual gland.     

Ultrastructural and histochemical study of the salivary glands of Aplysia depilans (Mollusca, Opisthobranchia)

The digestive system of the sea hare, Aplysia depilans , includes a pair of ribbon-shaped salivary glands. A central duct and a large blood vessel run close to each other along the length of these glands and both are surrounded by a layer of muscle cells. Three cell types form the glandular epithelium: granular cells, vacuolated cells and mucocytes. The granular cells possess cilia and spherical secretion granules, located primarily in the apical region. The granules of immature cells have a low electron density and are mainly formed by neutral polysaccharides with small amounts of proteins. The granules of mature cells are larger, have a high electron density and are mainly formed by proteins with lower amounts of neutral polysaccharides. Transition stages between immature and mature granular cells are observed. The vacuolated cells are large and frequently pyramidal in shape, but after the application of histochemical techniques almost all vacuoles remain uncoloured. The numerous vacuoles contain flocculent material in a clear background and the mitochondria possess large crystalline structures in the matrix. A pyramidal shape is also typical of the mucocytes, which are filled with vesicles containing granular masses surrounded by a network of secretion material. These large cells are strongly stained by Alcian blue, revealing the presence of acidic mucopolysaccharides. This is the first ultrastructural study of the salivary glands in opisthobranch gastropods.     

Morpho-histochemical characterization of salivary gland cells of males of the tick <Emphasis Type="Italic">Rhipicephalus</Emphasis><Emphasis Type="Italic">sanguineus</Emphasis> (Acari: Ixodidae) at different feeding stages: description of new cell types

This study describes the changes undergone by cells of the salivary glands of unfed and feeding (at day two and four post-attachment) Rhipicephalus sanguineus males, as well as new cell types. In unfed males, types I and II acini are observed with cells “undifferentiated”, undefined 1 and 2 (the latter, with atypical granules), a, c1 and c3; type III is composed of cells d and e; and type IV present cells g. In males at day two post-attachment, type I acini exhibit the same morphology of unfed individuals. An increase in size is observed in types II, III, and IV, as cells are filled with secretion granules. Some granules are still undergoing maturation. In type II acinus, cells a, b and c1–c8 are observed. Cells c7 and c8 are described for the first time. Cells c7 are termed as such due to the addition of polysaccharides in the composition of the secretion granules (in unfed individuals, they are termed undefined 1). Type III acini exhibit cells d and e completely filled with granules, and in type IV, cells g contain granules in several stages of maturation. In males at day four post-attachment, type I acini do not exhibit changes. Granular acini exhibit cells with fewer secretion granules, which are already mature. In type II acini, cells a, b, c1–c5 are present, type III exhibit cells d and e, and type IV contain cells g with little or no secretion. This study shows that in the salivary glands of R. sanguineus males, cells a, c1, and c3 of type II acinus, and cells d and e of type III do not exhibit changes in granular content, remaining continuously active during the entire feeding period. This indicates that during the intervals among feeding stages, gland cells reacquire the same characteristics found in unfed individuals, suggesting that they undergo reprogramming to be active in the next cycle.     

Active participation of apoptosis and mitosis in sublingual gland regeneration of the rat following release from duct ligation

Summary This study was designed to establish how mitotic cell proliferation and apoptotic cell death participate in the regeneration of atrophied rat sublingual glands. To induce atrophy to the sublingual gland of rats, the excretory duct was ligated unilaterally near the hilum, and after 1 week of ligation (day 0) the duct ligation was released to enable gland regeneration. The regenerating glands were examined with routine histology, immunohistochemistry for proliferating cell nuclear antigen (PCNA) as a marker of proliferating cells, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) as a marker of apoptotic cells, and transmission electron microscopy. At day 0, a few acini and many ducts remained in the atrophic sublingual glands, and newly formed immature acini were observed at day 3. Thereafter acinar cells progressively matured and increased in number, although the number of ducts decreased. Many PCNA- and some TUNEL-positive cells were seen in acini and ducts during regeneration. The labeling indices for both cell types were statistically significantly different from that of the control at several time points of the regeneration. Apoptotic and mitotic cells were also confirmed to be present in the experimental sublingual glands by electron microscopy. These observations suggest that apoptosis as well as mitosis of duct and acinar cells actively participate in and play important roles in sublingual gland regeneration.     

Ultrastructural identification of the antennal gland complement in Siagona europaea Dejean 1826, a myrmecophagous carabid beetle

We examined antennal exocrine glands in adults of a myrmecophagous carabid beetle, Siagona europaea Dejean 1826 (Coleoptera, Carabidae), by light and electron microscopy and we identified two types of integumentary glands. The first type includes glands formed by three cells (a secretory cell, an intercalary cell and a duct cell) known as class 3 of Noirot and Quennedey (1991). The secretory cell has several large multivesicular electron‐lucent bodies, indicating a glycoprotein product associated with lipids. We hypothesize that this secretion protects the surface of antennae and sensilla from wear. The second group of glands includes unicellular glands known as oenocytes (class 2 of Noirot and Quennedey, 1991), which secrete epicuticular hydrocarbons through epidermal cells.     

Ultrastructural investigation of a salivary gland in a centipede: structure and origin of the maxilla I-gland of Scutigera coleoptrata (Chilopoda, Notostigmophora)

The maxilla I-gland of Scutigera coleoptrata was investigated using light and electron microscopy methods. This is the first ultrastructural investigation of a salivary gland in Chilopoda. The paired gland opens via the hypopharynx into the foregut and extends up to the third trunk segment. The gland is of irregular shape and consists of numerous acini consisting of several gland units. The secretion is released into an arborescent duct system. Each acinus consists of multiple of glandular units. The units are composed of three cell types: secretory cells, a single intermediary cell, and canal cells. The pear-shaped secretory cell is invaginated distally, forming an extracellular reservoir lined with microvilli, into which the secretion is released. The intermediary cell forms a conducting canal and connects the secretory cell with the canal cell. Proximally, the intermediary cell bears microvilli, whereas the distal part is covered with a distinct cuticle. The cuticle is a continuation of the cuticle of the canal cells. This investigation shows that the structure of the glandular units of the salivary maxilla I-gland is comparable to that of the glandular units of epidermal glands. Thus, it is likely that in Chilopoda salivary glands and epidermal glands share the same ground pattern. It is likely that in compound acinar glands a multiplication of secretory and duct cells has taken place, whereas the number of intermediary cells remains constant. The increase in the number of salivary acini leads to a shifting of the secretory elements away from the epidermis, deep into the head. Comparative investigations of the different head glands provide important characters for the reconstruction of myriapod phylogeny and the relationships of Myriapoda and Hexapoda.     

Localization and visualization of a coxiella-type symbiont within the lone star tick, Amblyomma americanum

A Coxiella-type microbe occurs at 100% frequency in all Amblyomma americanum ticks thus far tested. Using laboratory-reared ticks free of other microbes, we identified the Amblyomma-associated Coxiella microbe in several types of tissue and at various stages of the life cycle of A. americanum by 16S rRNA gene sequencing and diagnostic PCR. We visualized Amblyomma-associated Coxiella through the use of a diagnostic fluorescence in situ hybridization (FISH) assay supplemented with PCR-based detection, nucleic acid fluorescent staining, wide-field epifluorescence and confocal microscopy, and transmission electron microscopy (TEM). Specific fluorescent foci were observed in several tick tissues, including the midgut and the Malpighian tubules, but particularly bright signals were observed in the granular acini of salivary gland clusters and in both small and large oocytes. TEM confirmed intracellular bacterial structures in the same tissues. The presence of Amblyomma-associated Coxiella within oocytes is consistent with the vertical transmission of these endosymbionts. Further, the presence of the Amblyomma-associated Coxiella symbiont in other tissues such as salivary glands could potentially lead to interactions with horizontally acquired pathogens.     

Evolutionary regularities of somatic polyploidy in salivary glands of the Gastropod molluscs. II. Subclass Pectinibranchia: order Anisorbranchia

Salivary glands of 5 species of gastropod molluscs of the order Anisobranchia, the most ancient order within the subclass Pectinibranchia, have been investigated by histological and cytochemical methods, including DNA cytophotometry. Glandular cells of the following types were recognized: granular cells (with glycoproteid granular inclusions), mucocytes-I (including sulfatic acid mucopolysaccharides), mucocytes-II (including neutral and acid polysaccharides and proteins), and epithelial ciliated cells. All the described cell types are considered to be independent and their morphofunctional characteristics coincide with those of salivary gland cells of the gastropod molluscs of subclasses Cyclobranchia and Scutibranchia. It has been shown that somatic polyploidy in salivary glands in the Anisobranchia molluscs, likely as in those of other Archaeogastropoda (Cyclobranchia and Scutibranchia), is actually absent.     

Receptors for the neuropeptides,myoinhibitory peptide and SIFamide,in control of the salivary glands of the blacklegged tick Ixodes scapularis

Tick salivary glands are important organs that enable the hematophagous feeding of the tick. We previously described the innervation of the salivary gland acini types II and III by a pair of protocerebral salivary gland neurons that produce both myoinhibitory peptide (MIP) and SIFamide (?imo et al., 2009b). In this study we identified authentic receptors expressed in the salivary glands for these neuropeptides. Homology-based searches for these receptors in the Ixodes scapularis genome sequence were followed by gene cloning and functional expression of the receptors. Both receptors were activated by low nanomolar concentrations of their respective ligands. The temporal expression patterns of the two ligands and their respective receptors suggest that the SIFamide signaling system pre-exists in unfed salivary glands, while the MIP system is activated upon initiation of feeding. Immunoreactivity for the SIFamide receptor in the salivary gland was detected in acini types II and III, surrounding the acinar valve and extending to the basal region of the acinar lumen. The location of the SIFamide receptor in the salivary glands suggests three potential target cell types and their probable functions: myoepithelial cell that may function in the contraction of the acini and/or the control of the valve; large, basally located dopaminergic granular cells for regulation of paracrine dopamine; and neck cells that may be involved in the control of the acinar duct and its valve.     

Identification and subcellular localization of paracellin-1 (claudin-16) in human salivary glands

Salivary calcium plays an important role in the pathogenesis of dental caries and the bio-mineralization of dental enamel and exposed dentin. The cellular and molecular basis of calcium secretion by the human salivary glands is, however, poorly understood. Recently a transcellular transport of calcium by the acinus cells has been proposed. In this paper we looked for evidence for paracellular calcium transport by investigating the presence and cellular localization of paracellin-1 (claudin-16) that has been implied in paracellular magnesium and calcium transport in the kidney. At the mRNA level, using RT-PCR with primers of appropriate sequence, paracellin-1 mRNA could be found in human Glandula parotis, Glandula submandibularis, Glandula labialis and Glandula sublingualis samples. In addition, a splice variant was detected in three out of 15 glands consisting of exons one and five of the paracellin gene. In immunohistochemical studies paracellin-1 colocalised in the salivary excretory ducts with the tight junction proteins ZO-1 and occludin suggesting a potential role in paracellular calcium and magnesium transport. In the acini no such colocalisation was observed; paracellin was instead detected at the basal poles of the cells, between cells of the same acinus as well as between cells of neighboring acini. At this location paracellin-1 might act as selectivity filter for the paracellular movement of ions and water during stimulated secretion. Thus, both in the ducts and in the acini a paracellular transport of calcium appears possible. Whether it occurs at all and the extent to which it contributes to the overall salivary calcium secretion remains, however, to be determined.     

Early markers of regeneration following ductal ligation in rat submandibular gland

Rat submandibular glands can recover their function and secretory protein content following ductal ligation-induced atrophy. Morphological studies have established that following ligation, deligation of the gland allows the regeneration of new salivary gland tissue. However, little is known about changes happening during early regeneration following intra-oral duct ligation, which does not damage the parasympathetic nerves. Glands that had been 2 weeks ligated or 2 weeks ligated + 3 days deligated were compared. Tissue was prepared for histological, immunohistochemical (SMG-B and Ki-67) and immunocytochemical analyses (smooth muscle actin, aquaporin 5). Haematoxylin and eosin staining of deligated glands showed that some acini regained their cytoplasmic volume; moreover, the loss of Alcian blue/periodic acid-Schiff’s staining from the lumen of ducts suggested successful deligation. The deligated gland was characterized by atypical acinar-ductal branched structures, which were less frequent in the ligated gland and rarely seen in normal unoperated tissue. Myoepithelial cells were also investigated since changes in their morphology reflected changes in the acini morphology not readily detected by conventional staining. Actin staining revealed the presence of some shrunken acini in the atrophic tissue, whereas they had regained their normal morphology in the deligated gland suggesting that the acini were recovering. Some acini during deligation regained aquaporin 5 expression, which had decreased during atrophy. SMG-B protein, located in the pro-acinar cell during gland development and usually found in the intercalated duct cells in the adult, was detected in the newly formed acini of the deligated gland. This study suggests that morphological markers of regeneration appear as early as 3 days following ligation removal. The authors thank the Wellcome Trust for funding.     

Morphofunctional studies on human labial salivary glands

In this study, the first experimental investigation carried out at the ultrastructural level on mucous cells of human salivary glands, we have examined by light microscopy (LM), transmission electron microscopy (TEM), high resolution scanning electron microscopy (HRSEM), the secretory response of labial glands stimulated in vitro by the beta-adrenergic agent, D,L isoproterenol, and by the muscarinic agent carbachol. For comparison we have used identical methods to study samples of mixed portions of human submandibular glands. Morphological findings obtained here on both submandibular and labial glands mucous cells demonstrate that mucous droplets are released solely by muscarinic stimulation, and that cytological events occurring during secretory discharge are similar to those described by others, using TEM, on stimulated mucous cells of rat sublingual glands. Despite the fact that human labial glands are said to have a prominent cholinergic innervation with scanty adrenergic nerves, the response of seromucous cells in these organs to stimulation with carbachol and with isoproterenol was similar to that observed by us, (using LM, TEM and HRSEM), in serous cells of human major salivary glands.     

Biological behavior of myoepithelial cells in the regeneration of rat atrophied sublingual glands following release from duct ligation

Summary The present study aimed to clarify how myoepithelial cells behave during regeneration of an atrophied sublingual gland by investigating cell proliferation and ultrastructure. Atrophy of rat sublingual glands was induced by unilateral ligation of the excretory duct near the hilum with metal clips, which were then removed after one week of ligation for regeneration. The sublingual glands 0–14 days after unligation were examined with single immunohistochemistry for actin as a marker of myoepithelial cells, double immunohistochemistry for actin and proliferating cell nuclear antigen (PCNA) as a marker of proliferating cells, and transmission electron microscopy (TEM). The single immunohistochemistry and TEM showed that myoepithelial cells surrounded residual ducts in the atrophied glands and immature and mature acini in the regenerating glands. Although PCNA-positive myoepithelial cells were identified during regeneration, PCNA labeling indices of myoepithelial cells were low at all time points except at day 7. Ultrastructurally, myoepithelial cells showing bizarre shaped structures in the atrophy changed with maturation of differentiating acinar cells and appeared normal in the regenerated glands. There was no differentiation of the remaining duct cells to myoepithelial cells. These observations suggest that proliferation of myoepithelial cells and differentiation to myoepithelial cells do not commonly participate in the regeneration of atrophied sublingual glands and that the bizarre shaped myoepithelial cells in the atrophied sublingual glands recover the original shapes with acinar cell regeneration.     

Amblyomma cajennense (Acari: Ixodidae): salivary gland cells of partially engorged females ticks and the production of lipid by their mitochondria

Morphologically, the salivary glands of ticks are paired structures consisting of a secretory and an excretory portion, lacking a reservoir for the storage of the secretion. The secretory portion is composed in females by cells that form acini classified into the types I, II, and III. The excretory possess a major duct, from which arise several intermediate ducts that then subdivide to form the canaliculi or acinal tubules, which end at the acini from where they collect the secretion. The present study describes the ultrastructural changes that occur in the mitochondria of cells of the acini I, II, and III in the salivary glands of partially engorged females of the Cayenne tick Amblyomma cajennense. The results show that this organelle exhibits completely disarrayed crests due to the presence of lipidic material inside the matrix and between the crests, thus demonstrating their participation in the production of the lipids that would be used structurally by the cells. These organelles with ultrastructural changes were denominated derived mitochondria.     

Evidence that developmental changes in type III acini in the tickAmblyomma hebraeum (Acari: Ixodidae) are initiated by a hemolymph-borne factor

During feeding, certain cells in the salivary gland type III acini of the ixodid tickAmblyomma hebraeum Koch undergo major developmental changes. We induced many of these changes in the ablumenal interstitial cells (AbIC), adlumenal interstitial cell (AdIC), and f-cells of type III acini, by transplanting the salivary gland of the unfed female to the hemocoel of a feeding female. In transplants, AbICs enlarged and formed a labyrinth of extracellular spaces. Extensions of AbICs pushed into the AdIC. Autophagic vacuoles were common in AbICs. The f-cells also enlarged and developed autophagic vacuoles. Complex interdigitation occurred between the f-cells and the AbIC. In transplants, the labyrinth was not as extensive as that of fed unoperated females or of operated females. The AdIC, AbIC, and f-cells did not undergo as extensive a development in unoperated fed males as the same cells did in unoperated fed females. In males AbICs did not develop an extensive labyrinth, and the f-cells did not develop beyond a secretory phase. No autophagic vacuoles were observed in any of these cells. When male salivary glands were transplanted into feeding females, AdIC, AbICs and f-cells developed an ultrastructure similar to the same cells in female transplants. Cells from salivary glands of unfed females cultured for 2 days in TC medium 199 resembled the same cells from control unfed salivary glands. The selectivity of these changes supports the conclusion that a hemolymph-borne salivary gland development factor initiated this development.     

Death by apoptosis in salivary glands of females of the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae)

The salivary glands of females of the tick Rhipicephalus sanguineus at three feeding stages: unfed, engorged, and at day three post-engorgement, were subjected to cytochemical methods of enzymatic analysis and cell viability. Comparing glands at these stages, was observed distinct staining patterns in cells of different types of acini, specially in degenerating types III, II, I, which were affected in this sequence by cell death. This study also revealed changes in: nuclei, staining intensity for acid phosphatase and ATPase activities, and permeability of the plasma membrane. Acid phosphatase activity was inversely proportional to that of ATPase, while ATPase activity was always proportional to membrane integrity. The glands of unfed females exhibited high metabolic activity and cells with intact nucleus and plasma membrane, suggesting that the presence of acid phosphatase detected in these individuals may participate in the normal physiology of some acini, as they were not undergoing degeneration. In acini I and II of engorged females, we observed cells with intact membranes, as well as changes characterized by nuclear changes, decrease in ATPase activity, and stronger acid phosphatase activity. At day three post-engorgement, degeneration progressed to more advanced stages, loss of membrane integrity was observed in most cells (of some type I acini, most type II acini, and all type III acini), as well as prominent nuclear changes, decrease in ATPase activity, and intense acid phosphatase activity, resulting in apoptotic bodies. During the death of cells nuclear changes preceded cytoplasmic ones in the following sequence: nuclear changes, loss of ATPase activity, loss of integrity of the plasma membrane, increase in acid phosphatase activity, and formation of apoptotic bodies. The presence of acid phosphatase with a secondary role (late) during cell death, degrading final cell remnants, characterized this process in the glands of R. sanguineus females as atypical or non-classic apoptosis.     

Evolutionary regularities of somatic polyploidy manifestation in salivary glands of gastropod molluscs. I. Subclasses Cyclobranchia and Scutibranchia

By means of histological and cytochemical methods, including DNA cytophotometry, the salivary glands of 11 species of molluscs of two old gastropod subclasses--Cyclobranchia and Scutibranchia (limpets)--have been investigated. In spite of some anatomical differences, the glandular epithelium of investigated molluscs includes functionally similar cell types: granular cells (with glycoproteid granular inclusions), mucocytes-I (that include sulfatic acid mucopolysaccharides), mucocytes-II (that include neutral and acid polysaccharides and proteins) and also the epithelial ciliated cells. Data of experiments on starvation and synchronous feeding of molluscs testify that all described cell types are independent. According to DNA cytophotometry data, the glandular cell nuclei are diploid in the main; only small part of the nuclei, varying in different species from 0.5 to 5.0%, displayed tetraploid DNA mass. A conclusion is made that in the oldest subclasses of gastropods (Cyclobranchia and Scutibranchia) somatic polyploidy, as a factor of tissue growth in salivary glands, is actually absent.     

Silk formation mechanisms in the larval salivary glands of<Emphasis Type="Italic">Apis mellifera</Emphasis> (Hymenoptera: Apidae)

The mechanism of silk formation inApis mellifera salivary glands, during the 5th instar, was studied. Larval salivary glands were dissected and prepared for light and polarized light microscopy, as well as for scanning and transmission electron microscopy. The results showed that silk formation starts at the middle of the 5th instar and finishes at the end of the same instar. This process begins in the distal secretory portion of the gland, going towards the proximal secretory portion; and from the periphery to the center of the gland lumen. The silk proteins are released from the secretory cells as a homogeneous substance that polymerizes in the lumen to form compact birefringent tactoids. Secondly, the water absorption from the lumen secretion, carried out by secretory and duct cells, promotes aggregation of the tactoids that form a spiral-shape filament with a zigzag pattern. This pattern is also the results of the silk compression in the gland lumen and represents a high concentration of macromolecularly well-oriented silk proteins.