Programmed cell death in the larval salivary glands of <Emphasis Type="Italic">Apis mellifera</Emphasis> (Hymenoptera,Apidae)

The morphological and histochemical features of degeneration in honeybee (Apis mellifera) salivary glands were investigated in 5th instar larvae and in the pre-pupal period. The distribution and activity patterns of acid phosphatase enzyme were also analysed. As a routine, the larval salivary glands were fixed and processed for light microscopy and transmission electron microscopy. Tissue sections were subsequently stained with haematoxylin-eosin, bromophenol blue, silver, or a variant of the critical electrolyte concentration (CEC) method. Ultrathin sections were contrasted with uranyl acetate and lead citrate. Glands were processed for the histochemical and cytochemical localization of acid phosphatase, as well as biochemical assay to detect its activity pattern. Acid phosphatase activity was histochemically detected in all the salivary glands analysed. The cytochemical results showed acid phosphatase in vesicles, Golgi apparatus and lysosomes during the secretory phase and, additionally, in autophagic structures and luminal secretion during the degenerative phase. These findings were in agreement with the biochemical assay. At the end of the 5th instar, the glandular cells had a vacuolated cytoplasm and pyknotic nuclei, and epithelial cells were shed into the glandular lumen. The transition phase from the 5th instar to the pre-pupal period was characterized by intense vacuolation of the basal cytoplasm and release of parts of the cytoplasm into the lumen by apical blebbing; these blebs contained cytoplasmic RNA, rough endoplasmic reticule and, occasionally, nuclear material. In the pre-pupal phase, the glandular epithelium showed progressive degeneration so that at the end of this phase only nuclei and remnants of the cytoplasm were observed. The nuclei were pyknotic, with peripheral chromatin and blebs. The gland remained in the haemolymph and was recycled during metamorphosis. The programmed cell death in this gland represented a morphological form intermediate between apoptosis and autophagy.     

Macromolecular array patterns of silk gland secretion in social Hymenoptera larvae

The cocoon, produced by most holometabolous insects, is built with silk that is usually produced by the larval salivary gland. Although this silk has been widely studied in the Lepidoptera, its composition and macromolecular arrangement remains unknown in the Hymenoptera. The macromolecular array patterns of the silk in the larval salivary gland of some meliponids, wasps, and ants were analyzed with polarized-light microscopy, and they were compared with those of Bombyx mori (Lepidoptera). There is a birefringent secretion in the glandular lumen of all larvae, due to filamentous structural proteins that display anisotropy. The silk in the distal, middle and proximal regions of the secretory portion of Formicidae and Vespidae glands presented a lattice optical pattern. We found a different pattern in the middle secretory portion of the Meliponini, with a zigzag rather than a lattice pattern. This indicates that the biopolymer fibers begin their macromolecular reorganization at this glandular region, different from the Formicidae and the Vespidae, in which the zigzag optical pattern was only found at the lateral duct. Probably, the mechanism of silk production in the Hymenoptera is a characteristic inherited from a common ancestor of Vespoidea and Sphecoidea; the alterations in the pattern observed in the Meliponini could be a derived characteristic in the Hymenoptera. We found no similarity in the macromolecular reorganization patterns of the silk between the Hymenoptera species and the silkworm.     

The post-embryonic changes in Melipona quadrifasciata anthidioides Lep. (Hym. Apoidea). II. Development of the salivary glands system

The paper deals with the development of the salivary gland system in Melipona quadrifasciata anthidioides, which begins in the prepupal stage. The silk glands degenerate by autolysis at the end of the larval stage. Degeneration is characterized by cytoplasmic vacuolization and pycnosis of the nuclei of the secretory cells. The glandular secretory portion of degenerated silk glands separates from the excretory ducts. The salivary glands develop from the duct of the larval silk glands. The thoracic salivary glands develop from the ducts of the secretory tubules and the head salivary glands from the terminal excretory duct. The mandibular glands appear in the prepupa as invaginations of mandibular segments, and their differentiation to attain the adult configuration occurs during pupation. The hypopharyngeal glands have their origin from evaginations of the ventral anterior portion of the pharynx. A long tubule first appears with walls formed by more than one cellular layer. Then some cells separate from the lumen of the duct, staying attached to it by a cuticular channel in part intracellular. The initial duct constitutes the axial duct, in which the channel of the secretory cells opens. During the development of salivary and mandibular glands, they recapitulate primitive stages of the phylogeny of the bees. During the development of salivary glands system, mitosis accounts for only part of the growth. Most of the growth occurs by increase in size of cells rather than by cell division. In brown-eyed and pigmented pupae six days before emergence, the salivary gland system is completely developed, although not yet functioning.     

Peculiar salivary glands in a silk‐producing mite Bakericheyla chanayi (Cheyletidae)

This is the first ultrastructural investigation of salivary glands in the family Cheyletidae. In both sexes of Bakericheyla chanayi, paired acinous salivary glands and tubular coxal glands were shown to be united into the common podocephalic system. The secretory portion of the salivary gland includes medial and lateral lobes composed of the five and two cells, respectively, with clearly distinct ultrastructure. The cytoplasm of the cells is occupied by the secretory granules containing fine fibrous material. The fine structure of both cell types suggest a proteinaceous nature of their secretions. A single central process extending from the apical face of each secretory cell passes through the common acinar cavity to enter the conducting duct. A pair of intercalary cells at the base of the conducting duct links it with the secretory portion of the gland. Extending towards the acinar cavity, protrusions of intercalary cells alternate the apical regions of the secretory cells and form with them highly‐specialized contacts characterized by the apical network of microtubules and microfilaments. Two possible ways of secretion are suggested: 1) exocytosis into the acinar cavity and 2) direct passage via the central processes. The detection of axon profiles in the gland body suggests a neural control for the glandular cell function. In tritonymphs, neither secretion nor large lateral lobe cells were observed up to the pharate stage when the lateral lobe undergoes rapid differentiation. The arrangement of the acinous gland is compared to that of other arthropods. Its composition appears to be close to the class three of insect glands. The involvement of the lateral lobe cells in silk production is discussed. J. Morphol. 276:772–786, 2015. © 2015 Wiley Periodicals, Inc.     

Ultrastructure of male accessory glands of Chrysomya megacephala (Fabricius) (Diptera: Calliphoridae)

The ultrastructure of the male accessory glands of the blow fly, Chrysomya megacephala (Fabricius), was presented using light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). A pair of accessory glands was separated at opposite sites. Morphometric results using LM yield evidenced no significant difference in the median of either length or width of the left and right glands. A significant increment in both length and width was seen to plateau between three to six days. SEM observation showed that the surface of the glands revealed a faint irregular groove pattern throughout, and it was occasionally penetrated by tracheoles. Each gland was a slender, elongated sac‐like tubule having apical rounded ends, with a slight constriction at the sub‐apical part of the gland being observed occasionally. TEM analyses of three‐day‐old males showed that the glands consisted of external capsular cells with a basement membrane underneath, glandular cells, and gland lumen. The capsular cell was flat and contained a nucleus with electron dense material in the nuclear envelope. The glandular cell, appearing as columnar, consisted of a vacuolated component that contained a large oval nucleus centrally or sub‐basally located, with dense mitochondria, numerous rough endoplasmic reticulum, and secretory vesicles containing electron‐lucent materials. In the gland lumen, the cross‐section through the middle portion revealed dense secretory materials, characterized by electron‐dense materials. Some sections revealed a large lumen where secretion accumulates within the delicate sac. The seven‐day‐old glands exhibited a remarkable change in the lumen, where the whole space contained a large amount of secretory materials, with the electron‐dense materials being characterized as similar to those observed in three‐day‐old glands. About four prominent types of secretions were observed on the basis of difference in electron‐density.     

Protein synthesis in the Chironomus thummi salivary gland

Summary Secretory proteins isolated from the lumen of the Chironomus thummi salivary gland were labelled with radioactive amino acids in vivo and in vitro. Under both conditions all but one of the electrophoretically separated fractions became labelled, the 6 prominent polypeptides already after 10–15 min of incubation. Differences in the labelling pattern during development from early 4th instar larvae to late prepupae were not detected.After synthesis the secretory proteins are stored in the cytoplasm for different times until they are exported into the gland lumen.None of the prominent protein fractions extracted only from the cells of the gland were found to be labelled even after labelling times up to 10 hrs. Therefore, it may be concluded that the Chironomus salivary gland synthesizes predominatly secretory proteins at least after the last larval moult.Long-time treatment of whole larvae with actinomycin D has no striking effect on the protein synthesis of the gland.Some of the results together with data from the literature led us to the speculation that changes of puff patterns (Balbiani rings excluded) do not reflect subsequent changes at the translational level.     

Secretory and basal cells of the epithelium of the tubular glands in the male Mullerian gland of the caecilian Uraeotyphlus narayani (Amphibia: Gymnophiona)

Caecilians are exceptional among the vertebrates in that males retain the Mullerian duct as a functional glandular structure. The Mullerian gland on each side is formed from a large number of tubular glands connecting to a central duct, which either connects to the urogenital duct or opens directly into the cloaca. The Mullerian gland is believed to secrete a substance to be added to the sperm during ejaculation. Thus, the Mullerian gland could function as a male accessory reproductive gland. Recently, we described the male Mullerian gland of Uraeotyphlus narayani using light and transmission electron microscopy (TEM) and histochemistry. The present TEM study reports that the secretory cells of both the tubular and basal portions of the tubular glands of the male Mullerian gland of this caecilian produce secretion granules in the same manner as do other glandular epithelial cells. The secretion granules are released in the form of structured granules into the lumen of the tubular glands, and such granules are traceable to the lumen of the central duct of the Mullerian gland. This is comparable to the situation prevailing in the epididymal epithelium of several reptiles. In the secretory cells of the basal portion of the tubular glands, mitochondria are intimately associated with fabrication of the secretion granules. The structural and functional organization of the epithelium of the basal portion of the tubular glands is complicated by the presence of basal cells. This study suggests the origin of the basal cells from peritubular tissue leukocytes. The study also indicates a role for the basal cells in acquiring secretion granules from the neighboring secretory cells and processing them into lipofuscin material in the context of regression of the Mullerian gland during the period of reproductive quiescence. In these respects the basal cells match those in the epithelial lining of the epididymis of amniotes.     

Immunocytochemical localization of seminal proteins in salivary and lacrimal glands of the rat

Antibodies against 10 different secretory proteins from the accessory sex glands of the male rat were used for immunohistochemical studies of salivary and lacrimal glands from intact and castrated rats, at the light- and electron-microscopic levels. In the parotid gland, secretory acinar cells showed immunoreactivity with antibodies against prostatic binding protein, cystatin-related peptide and acid phosphatase (isoenzyme pI 8.0; 5.6) typical of ventral prostate, and seminal vesicle secretion VI. Western blotting analysis indicated that immunoreactivity against prostatic binding protein was attributable to a subunit, presumably C3. Acid phosphatase pI 5.6 showed a molecular weight of 66 kDa, which is at variance with the prostatic form. Immunoreactivity for secretory transglutaminase, derived from the coagulating gland, was restricted to myoepithelial and stromal cells. In castrated animals, the immunoreactivity of acinar cells was reduced to the background level, whereas stromal transglutaminase immunoreactivity was unaltered. The distribution pattern of immunoreactivity for the proteins mentioned was almost identical in the lacrimal gland. Significant differences were however observed in the immunoreactivity of the inframandibular gland, where serous glandular cells were non-immunoreactive for seminal proteins, with the exception of acid phosphatase isoenzyme pI 8.0. Granules present in the convoluted granular ducts were immunoreactive particularly for acid phosphatase (isoenzyme pI 5.6)but much less for cystatin-related peptide; immunoreactivity was reduced after castration. The straight portion of the inframandibular duct system was immunoreactive for transglutaminase, but no influence of castration was visible. The distribution of immunoreactivity for seminal proteins present in the salivary and lacrimal glands and the pronounced androgen-dependence of their expression point to functional relationships of the respective proteins at both glandular sites.     

Influence of thyroid hormones on neuronal death and differentiation in larval Rana pipiens.

The sphingid moth, Manduca sexta, typically passes through five larval instars, a pupal, and an adult stage. The larval labial glands secrete silk in the first instar and a viscous lubricant in the fifth. During metamorphosis the glands develop into salivary organs which produce an invertase-rich secretion. In normal development, the uniform population of cells in the duct of the larval gland transforms into the four sequentially arranged regions of secretory and conductive cells of the adult gland. In order to determine when competence to form the adult gland is established, fragments of labial gland ducts from first through fifth instar larvae were implanted into pupae. These gland fragments underwent metamorphosis with their hosts, passing through the same developmental phases. Glands from as early as the first instar were competent to form histologically and functionally normal adult regions. In later instars, transplants of measured fragments demonstrated that larval cells were programmed in situ to develop into the four adult cell types.     

Immunofluorescence-microscopic demonstration of myosin and actin in salivary glands and exocrine pancreas of the rat

Summary Actin and myosin were localized in various salivary glands (parotid, submandibular, sublingual, lingual and Harderian gland) and the exocrine pancreas of rats by indirect immunofluorescence microscopy using specific rabbit antibodies against chicken gizzard myosin and actin. A bright immunofluorescent staining with both antibodies was observed at three main sites: (1) In myoepithelial cells of all salivary glands, (2) in secretory gland cells underneath the cell membrane bordering the acinar lumen (except Harderian and mucous lingual gland), and (3) in epithelial cells of the various secretory ducts (of all glands) in similar distribution as in acinar cells. The present immunohistochemical findings in acinar cells could lend further support to a concept suggesting that myosin and actin are involved in the process of transport and exocytosis of secretory granules.Supported by grants form Deutsche Forschungsgemeinschaft (Dr. 91/1, Ste. 105/19 and U. 34/4). We thank Mrs. Ursula König, Mrs. Christine Mahlmeister and Miss Renate Steffens for excellent technical assistance.     

Analysis of nuclear proteins from silk glands ofBombyx mori

A gentle method for the isolation of nuclei from developing silk glands ofBombyx mori has been standardized. The nuclei, whether isolated or directly visualizedin situ within the silk glands, exhibit complex morphology. The nuclei occupy almost the entire volume of the gigantic silk gland cells. Although the isolated nuclei still retain their ramified morphology, being polyploid they are fragile and often become fragmented. The histone and low-salt-extractable proteins from nuclei isolated from the middle and posterior silk glands on different days of the fourth and fifth instars of larval development have been analysed. The histones did not show any stage- or tissue-specific variations whereas the low-salt-extractable proteins showed some developmental stage specific variation. Using the antibody raised against one such protein, its absence in the early stage of development has been confirmed by Western blotting techniques. This developmental stage specific protein may be functionally linked to some activities responsible for boosting up the production of silk or silk-related proteins during the fifth instar of larval development.     

Fine structure of the silk gland in the mite Ornithocheyletia sp. (Prostigmata,Cheyletidae)

Silk spinning is widely-spread in trombidiform mites, yet scarse information is available on the morphology of their silk glands. Thus this study describes the fine structure of the prosomal silk glands in a small parasitic mite, Ornithocheyletia sp. (Cheyletidae). These are paired acinous glands incorporated into the podocephalic system, as typical of the order. Combined secretion of the coxal and silk glands is released at the tip of the gnathosoma. Data obtained show Ornithocheyletia silk gland belonging to the class 3 arthropod exocrine gland. Each gland is composed of seven pyramidal secretory cells and one ring-folded intercalary cell, rich in microtubules. The fine structure of the secretory cells points to intensive protein synthesis resulted in the presence of abundant uniform secretory granules. Fibrous content of the granules is always subdivided into several zones of two electron densities. The granules periodically discharge into the acinar cavity by means of exocytosis. The intercalary cell extends from the base of the excretory duct and contributes the wall of the acinar cavity encircling the apical margins of the secretory cells. The distal apical surface of the intercalary cell is covered with a thin cuticle resembling that of the corresponding cells in some acarine and myriapod glands. Axon endings form regular synaptic structures on the body of the intercalary cell implying nerve regulation of the gland activity.     

Morphological changes in the cephalic salivary glands of females and males of <Emphasis Type="Italic">Apis mellifera</Emphasis> and <Emphasis Type="Italic">Scaptotrigona postica</Emphasis> (Hymenoptera,Apidae)

The cephalic salivary glands of some species of bees are exclusive and well developed only in Apinae. These glands were studied with light and scanning electron microscopy in workers, queens and males from the honey bee Apis mellifera, and the stingless bee Scaptotrigona postica in different life phases. The results show that the cephalic salivary glands are present in females of both the species, and in males of S. postica. Nevertheless, they are poorly developed in young males of A. mellifera. In both species, gland growth is progressive from the time of emergence to the oldest age but, in A. mellifera males, the gland degenerates with age. Scanning electron microscopy shows that the secretory units of newly emerged workers are collapsed while in older workers they are turgid. Some pits on the surface of the secretory units correspond to open intercellular spaces. The possible functions of these glands in females and males of both species are discussed.     

Snythesis of mucoprotein secretion in the salivary glands of Drosophila melanogaster IIIrd instar larvae]

The process of mucoprotein (glue) accumulation is described. This substance is secreted by salivary glands of the III instar larvae of Drosophila melanogaster. Granules of the secretory substance appear in the glandular cells 90 hrs after egg laying. Its secretion takes place within 120 hrs. The template RNA of glue proteins appears to be synthesized within the range of 72-85 hrs since actinomycin treatment of larvae at this time suppresses the formation of secretory substance. The portion of secretory proteins amounts to 23-32% of the total gland protein. In a mutant, 13tl, deficient by the absence of pupation, no PAS-positive, staining of glands or secretory granules identified under the phase-contrast microscope was found. A possible participation of fat body in the formation of mucoprotein secretory substance is discussed.     

NEUROANATOMICAL STUDIES OF PROTHORACIC GLANDS OF COTTON BOLLWORM HELZCOVERPA ARMZCERA (LEPIDOPTERA: NOCTUIDAE)*

Abstract Gross anatomy, ultrastructure, innervation and ultrastructural alterations of the prothoracic gland (PTG) of cotton bollworm, Helicover pa armigera (Lepidoptera: Noctuidae) are illustrated for the last larval and early pupal stages as observed by light and electron microscopy. The T-shaped, paired (PTGs) consist each of 76–116 cells which are classified morphologically as large and small gland cells. In addition, another kind of small (about 6μ in diameter) gland cell was found in the PTGs of last instar larvae. The PTGs are innervated by the branches of 3 nerves! and tracheae and tracheoles are abundantly distributed to these glands. PTGs disappeared completely by the third day after ecdysis to the pupal stage (at temperature 28 C with a photoperiod L15^:D9). An intercellular channel system (ICS) is formed by numerous, deep invaginations of the plasma membrane of gland cells. This ICS gradually increases in depth and width and reaches maximum development around the time of the major ecdysteroid secretion peak during the last larval instar. Numerous multivesicular sacs (MVS) with their remnants and an extensive rough endoplasmic reticulum were observed within ICS and cytoplasm, respectively, on the fourth day of the last larval instar. At that time the matrix of mitochondria became much more electron lucent. Freeze-fracture replicas of the glandular epithelium were made from last instar (4th day larvae. Dynamics of structure are related to data from others concerning secretory states of the prothoracic glands of this species.     

Alteration of tubuliform silk gland cytoarchitecture with the reproductive cycle of the Western black widow spider,Latrodectus hesperus

The protein synthetic and secretory activity of spider tubuliform glands is known to be coordinated with the reproductive stage of the spider. For spiders that produce multiple egg cases, such as the black widow Latrodectus hesperus, this means that the cells that make up the tubuliform gland cycle from minimal to maximal silk protein synthesis and exocytosis as the spider transitions from early vitellogenesis to a gravid state and back. The impact of these transitions on the cells that form the tubuliform gland has yet to be characterized. The entire tubuliform gland undergoes an elastic deformation, doubling in size in response to the accumulation and depletion of egg case silk proteins within its lumen. Similarly, the diversity and organization of organelles within the cytoplasm of the secretory epithelial cells that make up the wall of the tubuliform gland change with the reproductive stage of the spider. Progression of a spider from early to late vitellogenesis is accompanied by decondensed nucleoli and distention of the rough endoplasmic reticulum, markers of protein synthetic activity. The presumed silk proteins that fill the lumen of the tubuliform gland of a gravid spider include a fibrous matrix with homogeneous spherical inclusions. These components are also present within the cytoplasm of the cell; however, only the fibrous material appears to be enclosed by membranous organelles. Transition of the tubuliform gland from peak silk synthesis back to a quiescent state is marked by the appearance of multivesicular bodies and organelles resembling phagophores and autophagosomes, suggestive of a role for autophagy in the process of recovery. The reproducible cellular dynamics of the tubuliform silk gland of the black widow spider makes it a potential model system for study of the regulation of silk gene expression, endomembrane transport, and exocytosis of silk proteins and autophagy.     

The prepupal salivary glands of Drosophila melanogaster

Evidence is presented in support of the concept that the larval salivary gland of Drosophila melanogaster continues to function as an important secretory organ throughout prepupal stages and after pupation. Just after puparium formation, and at other later periods, the glands appear to be in the process of disintegration, but each time they recover until after pupation. Nuclear blebbing occurs through the time of survival of the glands, but is shown not to involve transport of RNA out of the nucleus. Transport in and out of the nucleus is clearly rapid and in a steady state as compared to the massive and intermittent export of cytoplasmic substance into the lumen of the gland.This work was supported by grants from the National Science Foundation (GB-23343, PCH-02044).     

Structure of the male reproductive accessory glands of Pterostichus nigrita (Coleoptera: Carabidae), their role in spermatophore formation

Accessory gland secretions of male insects have many important functions including the formation of spermatophores. We used light and electron microscopy to investigate the structure of the accessory glands and posterior vasa deferentia of the carabid beetle Pterostichus nigrita to try to determine where spermatophore material is produced. Each accessory gland and posterior vas deferens had an outer layer of longitudinal muscle, beneath which was a layer of connective tissue and a thin band of circular muscle, all of which surrounded a layer of epithelial cells lining the lumen of the ducts. Based on the ultrastructure of the epithelial cells, and their secretory products, we identified two epithelial cell types in each region (distal and proximal) of the accessory glands and four types in the posterior vas deferens. Most secretory products, which stained positively for proteins and some mucins, were released into the lumen of the ducts by apocrine secretion. The accessory glands produced one type of secretory product whereas in posterior vasa deferentia, four types of secretory products were found layered in the lumen. Our results suggest that most of the structural material used to construct a spermatophore is produced by the cells of the posterior vasa deferentia.     

Polarized secretion of an ectopic protein in Drosophila salivary glands in vivo.

Epithelial cells can secrete specific proteins in a polarized manner, either from the apical or basolateral surface. Intracellular protein sorting which results in polarized secretion has previously been studied using epithelial tissue culture cells. We describe here the use of Drosophila larval salivary glands for the study of polarized secretion by epithelia in vivo, and address whether an ectopically synthesized secretory protein can be sorted and targeted to the correct cell surface for secretion. Larval cuticle proteins (LCPs) and salivary gland secretion (Sgs) proteins of Drosophila melanogaster are apically secreted proteins that are produced respectively by the epidermis and salivary glands. We have transformed Drosophila with a hybrid gene consisting of the sgs-4 promoter sequence and the coding sequence for a variant (LCP-f2) of LCP-2. We have found that transgenic late third instar larvae produce LCP-f2 only in the salivary glands and that LCP-f2 is properly secreted in vivo in a polarized manner from only the apical surface of the cells into the gland lumen. The results indicate that apical secretion does not depend on a tissue-specific targeting signal contained within the protein.