Morphological and biochemical analyses of the salivary glands of the malaria vector, Anopheles darlingi.

Adult Anopheles darlingi salivary glands are paired organs located on either side of the esophagus. The male glands consist of a single small lobe. The female gland is composed of two lateral lobes, with distinct proximal and distal portions, and a medial lobe. The lobes are acinar structures, organized as a unicellular epithelium that surrounds a salivary canal. The general cellular architecture is similar among the lobes, with secretory material appearing as large masses that push the cellular structures to the periphery of the organ. Cells of the proximal-lateral lobes show asynchronous cycles of secretory activity and contain secretory masses with finely filamentous aspect. In the distal-lateral lobes, cells display synchronous cycles of activity, and have a dense secretory product with mottled pattern. Cells of the medial lobe have secretory masses uniformly stained and highly electrondense. Biochemical analysis of the adult female salivary glands revealed apyrase, alpha-glucosidase and lysozyme activities. Alpha-glucosidase and lysozyme activities are detected mostly in the proximal lobes while apyrase is mainly accumulated in the distal lobes. This differential distribution of the analyzed enzymes reflects a specialization of different regions for sugar and blood feeding. Thus, the morphological differences observed in the lobes correlate with functional ones.     

Regions of mosquito salivary glands distinguished by surface lectin-binding characteristics

Malaria sporozoites invade only the medial lobe and the distal portions of the lateral lobes of the salivary glands of vector mosquitoes. Because surface characteristics of these tissues may mark their identity, we determined whether particular lobes bear unique carbohydrates. A preliminary screen using fluorescein labeled lectins indicated that the salivary glands of female Aedes aegypti differed from other organs tested in their affinity for DBA but not UEA I. By means of electron microscopy, a panel of seven biotin-labeled lectins were used to describe the carbohydrates of the basal lamina. Binding of CON A and to a lesser extent RCA 120 was distributed over each lobe of the salivary glands. Other lectins, however, were specific to certain lobes. The lateral distal lobe bound CON A, DBA, PNA, RCA 120, UEAI and WGA. The lateral proximal lobes bound only CON A and DBA. In addition, the basal lamina possesses a net negative charge as indicated by the binding of cationic ferritin. Thus, the basal lamina over the various regions of the salivary glands is characterized by unique carbohydrates and this suggests that surface receptors may provide the specificity that limits invasion by sporozoites to particular lobes of the salivary glands.     

Die Bildung spezifischer Proteine während der Metamorphose der Speicheldrüsen von Wilhelmia lineata (Simuliidae)

Histolysis of pupal salivary glands and their further development during metamorphosis of Wilhelmia lineata were investigated by light microscopy. After pupal-adult apolysis the distal parts of the glands degenerate. The adult gland develops from a ring of tightly packed very small cells with small nuclei at the anterior end of the degenerating pupal gland. The gland of the female pharate adult consists of a spherical accessory gland with small cells and oval nuclei and a little U-shaped main gland with larger cells and round nuclei. The two U-shaped lobes are orientated horizontally. The gland of the male pharate adult has the same shape and cellular structure as the accessory gland of the female. During further development the main gland becomes greatly elongated as the cells increase in size, the cells become flattened, and the secretion of saliva into the gland lumen can be seen. The glands are fully differentiated at the time of pupal-adult ecdysis. Soluble proteins from the one-day-old female pharate adult show a very similar pattern to those of pupal and male glands. With growth of the female main gland and secretion of saliva into the gland lumen increasing amounts of proteins, which have been characterized earlier (Poehling et al., 1976) occur in the gland. This could suggest a specific rôle of these proteins during bloodsucking in the adult females.     

Comparative ultrastructure of the salivary glands of two phytopathogen vectors,the beet leafhopper,Circulifer tenellus (Baker), and the corn leafhopper,Dalbulus maidis DeLong and Wolcott (Homoptera : Cicadellidae)

The salivary glands of 2 leafhoppers, Circulifer tenellus and Dalbulus maidis (Homoptera : Cicadellidae) were examined by light and electron microscopy. Centrally located and occupying both the head and thorax, the salivary glands consist of 2 paired parts, the accessory glands and the principal glands. In C. tenellus and D. maidis, the accessory glands are large, multicelled lobes that lie anterior to the principal gland. They join the principal glands near the common salivary duct-gland junction via a thinner tubular duct. The principal glands of both species consist of large binucleate cells that differ in cytology and arrangement. These cells are easily distinguished by unique staining characteristics. Circulifer tenellus salivary gland cells are arranged in 2 lobes, the anterior lobe, made up of 3 concentric rings around the salivary duct and the posterior lobe, arranged in a loose pyramid extending above the foregut. Dalbulus maidis glands are similarly organized around the salivary duct.     

Cytoskeletal F-actin patterns in whole-mounted larval and adult salivary glands of the fleshfly, Sarcophaga bullata.

The patterns of filamentous actin were analysed in different larval, pupal and adult stages in the salivary glands of the fleshfly Sarcophaga bullata. Using the rhodamine labelled phalloidin staining method in combination with detergent extraction specific actin filament distribution was detected. The salivary glands which are histolysed during the process of metamorphosis show distinct cellular morphology and actin filament patterns in larvae and adults. The large third instar larval salivary gland cells contain a well developed apicolateral microvillar zone. In third instar larvae this microvillar zone invaginates and expands in the basal part of the lateral membranes. Larval salivary gland cells also contain numerous parallel basal actin bundles. The larval glands are histolysed during metamorphosis and adult glands are formed out of the imaginal cell group. At the onset of metamorphosis these basal actin bundles form a network of crossing bundles. The filamentous actin patterns of the proximal part of adult gland cells is confined to the apicolateral microvillar membranes. The cells in the distal, tubular part of the adult salivary glands show intense staining of their folded lateral membranes.     

Morphology of the anterior digestive system of tonnoideans (Gastropoda: Caenogastropoda) with an emphasis on the foregut glands

ABSTRACT

Tonnoideans are marine carnivorous caenogastropods that prey on different invertebrates, namely polychaetes, sipunculids, bivalve and gastropod molluscs, and echinoderms. The morphology of the digestive system of 20 species from five families of the Tonnoidea was examined (for most of these species for the first time), and the salivary glands of six of them were studied using serial histological sections. Most of the studied families are rather similar anatomically, except Personidae (Distorsio), which differs both in proboscis morphology and the structure of the salivary glands. In most tonnoideans the salivary glands are split morphologically and functionally into anterior and posterior lobes, the latter synthesising strong sulfuric acid. The ducts of the posterior lobes are lined with non-ciliated epithelium and receive usually paired ciliated ducts from the anterior lobes to form a non-ciliated common duct, opening into the buccal cavity. In Personidae, the salivary glands are not separated into lobes, but are instead composed of ramifying tubules that are histologically different in the proximal and distal parts. Radulae of Tonnoidea are rather variable, with different patterns of interlocking teeth, both in the transverse and longitudinal rows, which may be related to particular feeding mechanisms. Due to the peculiarities of Personidae, the close relationship between that family and the rest of the Tonnoidea is questioned.     

Human salivary gland branching morphogenesis: morphological localization of claudins and its parallel relation with developmental stages revealed by expression of cytoskeleton and secretion markers

Development of salivary glands is a highly complex and dynamic process termed branching morphogenesis, where branched structures differentiate into mature glands. Tight junctions (TJ) are thought to play critical roles in physiological functions of tubular organs, contributing to cell polarity and preventing lateral movement of membrane proteins. Evidence demonstrated that claudins are directly involved in TJ formation and function. Using immunohistochemistry and immunofluorescence we have mapped the distribution of claudins-1, 2, 3, 4, 5, 7 and 11 and compared it with the expression of differentiation markers in human salivary glands obtained from foetuses ranging from weeks 4 to 24 of gestation. Expression of all claudins, except claudin-2 was detected in the various phases of human salivary gland development, up to fully mature salivary gland. The expression of all claudins increased according to the progression of salivary gland maturation evidenced by the classical markers-cytokeratin 14, cytokeratin low molecular weight, smooth muscle actin and human secretory component. Tight junction proteins-claudins appear to be important in the final shape and physiological functions of human salivary glands and are parallel related with markers of salivary gland differentiation.     

Apyrase and α-glucosidase in the salivary glands of Aedes albopictus

An apyrase and an α-glucosidase were detected in the salivary glands extracts of adult Aedes albopictus. The apyrase is a 61,000 Da secreted protein that hydrolyses ATP and ADP. This protein is synthesized in adults and is preferentially accumulated in the distal lateral lobes of the female salivary glands. The α-glucosidase is a secreted 67,000 Da protein. This enzyme is synthesized during adult life and accumulated in the proximal-lateral lobes of both males and females. The results are discussed and compared with data previously obtained with Aedes aegypti salivary glands.     

Salivary glands in Cicadidae (Hemiptera: Cicadoidea): comparative morphology, ultrastructure, and their phylogenetic significance

The present investigation provides information on gross morphology and ultrastructure of salivary glands of species in Cicadidae in detail. The structure of the salivary glands of 11 representative species from 10 genera belonging to three subfamilies of Cicadidae was studied using light microscopy and transmission electron microscopy. In the examined species, the salivary glands are paired structures, and each of which is comprised of a principal gland (pg) and an accessory gland (ag). The pg is divided into anterior and posterior lobes, and both of which consist of numerous long digitate lobules. The lobules at the base of the long digitate lobules of posterior lobe are greatly short; here, we named as “short lobules.” All the lobules vary in size, disposition, length, and shape. The anterior lobe and posterior lobes are connected by an anterior–posterior duct (apd). Two efferent salivary ducts (esd), derived from corresponding posterior lobes, fuse to form a short common duct which enters into the saliva syringe. The ag is composed of a greatly tortuous and folded accessory salivary tube, a gular gland (gg) constituting of several acini, and an accessory salivary duct (asd). The asd joins the esd at the place where the latter emergences. Constituents and arrangement of the salivary glands, the number and shape of the long digitate lobules in the anterior and posterior lobes, and the visibility of the apd were promising characters for the taxonomic and phylogenetic analysis of Cicadoidea. The variations of secretory granules in size, shape, and electron density in lobule cells of pg of Platypleura kaempferi probably indicating different materials are synthesized. The absence of the infoldings of basal plasma membrane in the basal area of the cells and the presence of electron-lucent vesicles in the cytoplasm of the gg cells of P. kaempferi might suggest that the secretions of gg are more watery.     

Development and ageing of the salivary glands of adult male Aedes aegypti (L.) and Aedes togoi (theobald) mosquitoes (Dipteria : Culcidae)

The salivary glands of adult male Aedes aegypti and Aedes togoi (Diptera : Culicidae), varying in age from less than 1 day after emergence to 42 days or 33 days respectively, were examined by light microscopy. Following emergence, the trilobed glands rapidly accumulate secretory product and attain their full size within about 48 hr. The amount of secretion in fully developed glands shows marked individual variation, but it is predominantly located in the posterior parts of the glands. Ageing changes begin to appear after about 8 days (A. aegypti) or 10–11 days (A. togoi), and although there is wide individual variation in their extent, in general, they become progressively more severe until at the age of 4–5 weeks all mosquitoes are substantially affected. Since other tissues, including ganglia, also regress with age, it is unlikely that salivary gland changes are the prime cause of death.     

Members of the salivary gland surface protein (SGS) family are major immunogenic components of mosquito saliva

Mosquitoes transmit Plasmodium and certain arboviruses during blood feeding, when they are injected along with saliva. Mosquito saliva interferes with the host's hemostasis and inflammation response and influences the transmission success of some pathogens. One family of mosquito salivary gland proteins, named SGS, is composed of large bacterial-type proteins that in Aedes aegypti were implicated as receptors for Plasmodium on the basal salivary gland surface. Here, we characterize the biology of two SGSs in the malaria mosquito, Anopheles gambiae, and demonstrate their involvement in blood feeding. Western blots and RT-PCR showed that Sgs4 and Sgs5 are produced exclusively in female salivary glands, that expression increases with age and after blood feeding, and that protein levels fluctuate in a circadian manner. Immunohistochemistry showed that SGSs are present in the acinar cells of the distal lateral lobes and in the salivary ducts of the proximal lobes. SDS-PAGE, Western blots, bite blots, and immunization via mosquito bites showed that SGSs are highly immunogenic and form major components of mosquito saliva. Last, Western and bioinformatic analyses suggest that SGSs are secreted via a non-classical pathway that involves cleavage into a 300-kDa soluble fragment and a smaller membrane-bound fragment. Combined, these data strongly suggest that SGSs play an important role in blood feeding. Together with their role in malaria transmission, we propose that SGSs could be used as markers of human exposure to mosquito bites and in the development of disease control strategies.     

A salivary gland-specific, maltase-like gene of the vector mosquito, Aedes aegypti

Genomic and cDNA clones of a gene expressed specifically in the salivary glands of adult Aedes aegypti have been isolated and sequenced. This gene encodes an abundant mRNA that is transcribed throughout the male salivary gland but only in the cells of the proximal lateral lobes of the female gland. The deduced protein has many basic amino acids, several possible sites for N-glycosylation, and displays striking similarities with the products of a yeast maltase gene and three previously unidentified genes from Drosophila melanogaster. We propose the name 'Maltase-like I' (MalI) to designate this gene. The presumed function of this gene product is to assist the mosquito in its sugar-feeding capabilities. The mosquito and fruitfly genes have similar structural features 5' to the protein coding regions, indicating that these genes may share common control mechanisms.     

Structure of male accessory glands of Bolivarius siculus (fischer) (Orthoptera,Tettigoniidae) and protein analysis of their secretions

In Tettigoniidae (Orthoptera), male reproductive accessory glands are involved in the construction of a two‐part spermatophore; one part, the spermatophylax, is devoid of sperm and considered a nuptial gift. The morphology, ultrastructure, and secretion protein content of the male reproductive accessory glands from Bolivarius siculus were investigated. Two main groups of gland tubules open into the ejaculatory duct: the “first‐order” glands, a number of large anterior tubules, and the “second‐order” glands, smaller and more numerous tubules positioned posteriorly. Along with a further subdivision of the gland tubules, we here describe for the first time an additional gland group, the intermediate tubules, which open between first and second‐order glands. The mesoderm‐derived epithelium of all glands is a single layer of microvillated cells, which can be either flattened or cylindric in the proximal or distal region of the same gland. Epithelial cells, very rich in RER and Golgi systems, produce secretions of both electron‐dense granules and globules or electron‐transparent material, discharged into the gland lumen by apocrine or merocrine mechanisms, respectively. With one exception, a unique electrophoresis protein profile was displayed by each of the gland types, paralleling their unique morphologies. To assess the contribution of different types of accessory glands to the construction of the spermatophore, the protein patterns of the gland secretions were compared with those of the extracts from the two parts of the spermatophore. All samples showed bands distributed in a wide range of molecular weight, including proteins of very low molecular mass. However, one major high molecular weight protein band (>180 kDa) is seen exclusively in extracts from the first‐order glands, and corresponds to an important protein component of the spermatophylax. J. Morphol., 2009. © 2009 Wiley‐Liss, Inc.     

Morphological aspects of Culex quinquefasciatus salivary glands

The salivary glands of Culex quinquefasciatus female mosquitoes are paired organs composed of two lateral lobes with proximal and distal secretory portions, and a medial lobe. All portions comprise a simple epithelium that surrounds a salivary duct. In the apical portion of the medial lobe, non-secretory cells strongly resemble cells involved in ion and water transport. The general architecture of the secretory portions is similar between lobes. The appearance of the secretory material and the morphological aspect of the apical cell membrane are the most distinctive features among the three secretory portions. Cells in the lateral proximal lobe display thin membrane projections extending into a translucent and finely filamentous secretory product. At the lateral distal portion, the apical cell membrane forms an intricate meshwork that encloses a dark secretory product. Medial lobe secretory cells also contain secretory cavities surrounded by intracytoplasmic vesicles, all containing a very dark and uniform product. Scattered cells holding numerous vacuoles, some of them containing a small and electron-dense granule eccentrically located and resembling those of the diffuse endocrine system, are frequently observed in the periphery of all secretory portions. Immunofluorescence assays revealed that the distal portion of the lateral lobes contains apyrase, an enzyme putatively responsible for platelet aggregation inhibition, diffusely distributed in the cell cytoplasm.     

A tentative initiation inhibitor of chromosomal heterogeneous RNA synthesis

The nucleoside analogue 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole inhibits labelling of chromosomal, high molecular weight RNA in the salivary gland cells of Chironomus tentans but does not interfere with the synthesis of ribosomal RNA and chromosomal low molecular weight RNA. When DRB2 was added after an initial labelling period (pulse-chase experiment) the radioactivity diminished preferentially in the lower molecular weight region of the HnRNA spectrum. After short chase periods the activity decreased moderately, or even increased, in the higher molecular weight region of the spectrum (75–100 S). After prolonged chases there was an overall and similar reduction in the activity in the whole HnRNA distribution. If the glands were preincubated in DRB for a short period before exposure to radioactive precursors, the label was again diminished more in HnRNA of low molecular weight than in that of higher molecular weight. When α-amanitin or actinomycin D, both known to be inhibitors of RNA chain elongation, replaced DRB in pulse-chase experiments, labelling of HnRNA was depressed in all size classes to the same extent. The accumulated data suggest that DRB acts, in explanted salivary gland cells, at the polymerase level by interfering with the initiation of chromosomal HnRNA synthesis.     

Morpho-functional variety of the coxal glands in cheyletoid mites (Prostigmata). II. Cheyletidae

Trombidiform mites are characterized by the presence of several paired glands in the anterior body portion united by a common conducting duct (podocephalic canal). Apart from the acinous (salivary) glands the podocephalic system includes a pair of tubular coxal glands (CGs) responsible for osmoregulation. The aim of the present study was to figure out how functional changes of acinous glands reflect on the corresponding CG. For this purpose, the anatomy and fine structure of the CG were analyzed in two mite species, Bakericheyla chanayi and Ornithocheyletia sp. (Cheyletidae), which have a different composition of their single acinous gland.The results showed that in both species the CG lacks a filtering saccule. It is composed of the proximal and distal tubes and leads into a cuticle-lined excretory duct. Both tubes demonstrate a similar species-specific fine structure. They are characterized by an extensive system of apical membrane invaginations (internal canals) associated with numerous large mitochondria. Local areas of modified internal canals were regularly observed in both species. They contain structures resembling those constituting filtering slit diaphragms of other animals.In O. sp., CG cells in addition demonstrate features characteristic of protein-like secretion. Apparently this correlates with the loss of true salivary glands in this species, as its acinous gland was previously assumed as silk producing. Contrary to this, the CG of B. chanayi shows no kind of granulation, which coincides with the presence of a salivary portion in its complex acinous gland.The microtubule-rich intercalary cells at the base of the excretory duct were associated with special muscles presumably regulating the dilation of the duct lumen. These cells might represent a basic feature common to different types of podocephalic glands.     

Peripheral cells in the salivary glands of female Aedes aegypti and A. togoi mosquitoes

A new type of cell, the peripheral cell, is described. These cells are located at the perimeter of the simple tubules which form the distal zones of the lateral lobes of the salivary glands of female Aedes aegypti and A. togoi. They may represent degenerate secretory cells which are segregated so that their altered secretory product cannot be discharged during blood feeding.     

Salivary amino acids in Lygus species (Heteroptera:Miridae)

Free and protein-bound amino acids were investigated in the phytophagous bug Lygus rugulipennis and its salivary gland. Over 38 substances were separated. The total content of amino compounds in the insects was about 1400 μmol/g fr. wt (16% by weight), of which 97% was amino acid residues in proteins.The salivary glands, which comprise about 1.5% of the live weight of the insects, contain 3.5% of the total free amino acids and 1% of the whote insect. Free and protein-bound amino acids comprise, respectively, about 1.4 and 11.6% of the fresh weight of the gland. The total concentration of free amino acids in the saliva was estimated to range from 0.5 to 2.2% by weight (ca. 0.1 M).The composition of free amino acids in the salivary gland of Lugus varies markedly. In four studied species (L. rugulipennis, L. gemellatus, L. pratensis, L. punctatus), the most abundant compounds were proline, arginine, lysine, leucine, glutamic acid, methionine sulphoxide and glycerophosphoethanolamine. In whole specimens of L. rugulipennis the predominant free amino acids were proline, alanine, taurine, glutamic acid, glutamine and methionine sulphoxide. The most abundant amino acids in proteins were glutamic and aspartic acid, glycine, alanine and leucine. The results indicate that the amino acid composition in the salivary glands of Lygus species does not differ markedly from that of the whole insect. The functions of salivary amino acids are discussed.