Characterization and description of a virus causing salivary gland hyperplasia in the housefly,Musca domestica

Abstract. A double-stranded DNA virus was isolated from hyperplasic salivary glands of male and female houseflies, Musca domestica L. (Diptera: Muscidae), collected from a dairy in Alachua County, Florida, U.S.A. Sodium dodecyl sulphate (SDS)–polyacrylamide gel electrophoresis (PAGE) of this housefly salivary gland hyperplasia (SGH) virus revealed the presence of two major and eight minor structural polypeptides. Restriction endonuclease analysis indicated that the c. 137 kilobase pair DNA was double-stranded. Weekly sweep-net sampling of the fly population throughout the season (May-October, 1991) showed that 1.5-18.5% of the dissected flies possessed hyperplasic salivary glands. The virus replicated within the nuclei of the salivary gland cells and was transmitted per os to newly-emerged healthy adult flies.     

Salivary gland hypertrophy virus of house flies in Denmark: prevalence, host range, and comparison with a Florida isolate

House flies (Musca domestica) infected with Musca domestica salivary gland hypertrophy virus (MdSGHV) were found in fly populations collected from 12 out of 18 Danish livestock farms that were surveyed in 2007 and 2008. Infection rates ranged from 0.5% to 5% and averaged 1.2%. None of the stable flies (Stomoxys calcitrans), rat-tail maggot flies (Eristalis tenax) or yellow dung flies (Scathophaga stercoraria) collected from MdSGHV-positive farms displayed characteristic salivary gland hypertrophy (SGH). In laboratory transmission tests, SGH symptoms were not observed in stable flies, flesh flies (Sarcophaga bullata), black dump flies (Hydrotaea aenescens), or face flies (Musca autumnalis) that were injected with MdSGHV from Danish house flies. However, in two species (stable fly and black dump fly), virus injection resulted in suppression of ovarian development similar to that observed in infected house flies, and injection of house flies with homogenates prepared from the salivary glands or ovaries of these species resulted in MdSGHV infection of the challenged house flies. Mortality of virus-injected stable flies was the highest among the five species tested. Virulence of Danish and Florida isolates of MdSGHV was similar with three virus delivery protocols, as a liquid food bait (in sucrose, milk, or blood), sprayed onto the flies in a Potter spray tower, or by immersiion in a crude homogenate of infected house flies. The most effective delivery system was immersion in a homogenate of ten infected flies/ml of water, resulting in 56.2% and 49.6% infection of the house flies challenged with the Danish and Florida strains, respectively.     

Disease dynamics and persistence of Musca domestica salivary gland hypertrophy virus infections in laboratory house fly (Musca domestica) populations

Past surveys of feral house fly populations have shown that Musca domestica salivary gland hypertrophy virus (MdSGHV) has a worldwide distribution, with an average prevalence varying between 0.5% and 10%. How this adult-specific virus persists in nature is unknown. In the present study, experiments were conducted to examine short-term transmission efficiency and long-term persistence of symptomatic MdSGHV infections in confined house fly populations. Average rates of disease transmission from virus-infected to healthy flies in small populations of 50 or 100 flies ranged from 3% to 24% and did not vary between three tested geographical strains that originated from different continents. Introduction of an initial proportion of 40% infected flies into fly populations did not result in epizootics. Instead, long-term observations demonstrated that MdSGHV infection levels declined over time, resulting in a 10% infection rate after passing through 10 filial generations. In all experiments, induced disease rates were significantly higher in male flies than in female flies and might be explained by male-specific behaviors that increased contact with viremic flies and/or virus-contaminated surfaces.     

Fine structure of the salivary glands of Triatoma infestans (Hemiptera: Reduviidae)

The fine structure of the salivary glands of adult Triatoma infestans (Hemiptera: Reduviidae) bugs has been analyzed. Stereomicroscopy and scanning electron microscopy showed that each insect presents a pair of salivary glands, each pair containing three distinct units (main, supplementary, and accessory) with different sizes and colors. Transmission electron microscopy demonstrated that all gland units consist of a monolayer of epithelial cells surrounding a large central lumen. The gland units are enveloped by a thick basal lamina containing bundles of muscle cells. Microvilli are present at the apical plasma membrane domain of the gland cells, thus enlarging the available membrane area for saliva secretion towards the large gland lumen, although occasionally budding vesicles could be observed among the microvilli. Cytochemical analysis showed that the salivary gland cells of T. infestans present abundant endoplasmic reticulum profiles and several lipid droplets.     

Par-1b is required for morphogenesis and differentiation of myoepithelial cells during salivary gland development

The salivary epithelium initiates as a solid mass of epithelial cells that are organized into a primary bud that undergoes morphogenesis and differentiation to yield bilayered acini consisting of interior secretory acinar cells that are surrounded by contractile myoepithelial cells in mature salivary glands. How the primary bud transitions into acini has not been previously documented. We document here that the outer epithelial cells subsequently undergo a vertical compression as they express smooth muscle α-actin and differentiate into myoepithelial cells. The outermost layer of polarized epithelial cells assemble and organize the basal deposition of basement membrane, which requires basal positioning of the polarity protein, Par-1b. Whether Par-1b is required for the vertical compression and differentiation of the myoepithelial cells is unknown. Following manipulation of Par-1b in salivary gland organ explants, Par-1b-inhibited explants showed both a reduced vertical compression of differentiating myoepithelial cells and reduced levels of smooth muscle α-actin. Rac1 knockdown and inhibition of Rac GTPase function also inhibited branching morphogenesis. Since Rac regulates cellular morphology, we investigated a contribution for Rac in myoepithelial cell differentiation. Inhibition of Rac GTPase activity showed a similar reduction in vertical compression and smooth muscle α-actin levels while decreasing the levels of Par-1b protein and altering its basal localization in the outer cells. Inhibition of ROCK, which is required for basal positioning of Par-1b, resulted in mislocalization of Par-1b and loss of vertical cellular compression, but did not significantly alter levels of smooth muscle α-actin in these cells. Overexpression of Par-1b in the presence of Rac inhibition restored basement membrane protein levels and localization. Our results indicate that the basal localization of Par-1b in the outer epithelial cells is required for myoepithelial cell compression, and Par-1b is required for myoepithelial differentiation, regardless of its localization.     

Localization of AQP5 during development of the mouse submandibular salivary gland

Aquaporin 5 (AQP5) is known to be central for salivary fluid secretion. A study of the temporal-spatial distribution of AQP5 during submandibular gland (SMG) development and in adult tissues might offer further clues to its unknown role during development. In the present work, SMGs from embryonic day (E) 14.5–18.5 and postnatal days (P) 0, 2, 5, 25, and 60 were immunostained for AQP5 and analyzed using light microscopy. Additional confocal and transmission electron microscopy were performed on P60 glands. Our results show that AQP5 expression first occurs in a scattered pattern in the late canalicular stage and becomes more prominent and organized in the terminal tubuli/pro-acinar cells towards birth. Additional apical membrane staining in the entire intralobular duct is found just prior to birth. During postnatal development, AQP5 is expressed in both the luminal and lateral membrane of pro-acinar/acinar cells. AQP5 is also detected in the basal membrane of acinar cells at P25 and P60. In the intercalated ducts at P60, the male glands show apical staining in the entire segment, while only the proximal region is positive in the female glands. These results demonstrate an evolving distribution of AQP5 during pre- and postnatal development in the mouse SMGs.     

Early membrane injury in lethally irradiated salivary gland cells

The early manifestations of radiation injury in salivary glands were investigated in the rat. The animals received a single X-ray dose in the range of 200-2000 rad to their neck area. Glandular changes during the first 24 hours were studied by light and electron microscopy and by measuring serum amylase activity. The amount of cell necrosis was quantitated and expressed as necrosis index (NI), Parotid NI and serum amylase activity 24 hours following irradiation were directly proportional to the X-ray dose. The submandibular gland cells were radioresistant and so were the mucous cells of the sublingual gland. The major increase in parotid acinar cell necrosis occurred between 12 and 24 hours after irradiation. However, more than 100 per cent increase in serum amylase level was detected prior to the onset of any significant cell necrosis. As early as two hours following irradiation signs of cell membrane injury were demonstrable in the parotid by electron microscopy and consisted of intracellular oedema, sequestered degenerative cell membranes, and an accumulation of intramitochondrial particles. None of these changes was detectable in the submandibular gland. The implication of membrane injury in the lethal effects of radiation on parotid cells is discussed.     

Identification of major soluble salivary gland proteins in teneral Glossina morsitans morsitans

Salivary glands of tsetse flies (Diptera: Glossinidiae) contain molecules that are involved in preventing blood clotting during feeding as well as molecules thought to be intimately associated with trypanosome development and maturation. Here we present a protein microchemical analysis of the major soluble proteins of the salivary glands of Glossina morsitans morsitans, an important vector of African trypanosomes. Differential solubilization of salivary proteins was followed by reverse-phase, high-performance liquid chromatography (HPLC) and analysis of fractions by 1-D gel electrophoresis to reveal four major proteins. Each protein was subjected to amino acid microanalysis and N-terminal microsequencing. A protein chemical approach using high-resolution 2-D gel electrophoresis and mass spectrometry was also used to identify the salivary proteins. Matrix-assisted, laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry and quadrupole time-of-flight (Q-TOF) tandem mass spectrometry methods were used for peptide mass mapping and sequencing, respectively. Sequence information and peptide mass maps queried against the NCBI non-redundant database confirmed the identity of the first protein as tsetse salivary gland growth factor-1 (TSGF-1). Two proteins with no known function were identified as tsetse salivary gland protein 1 (Tsal 1) and tsetse salivary gland protein 2 (Tsal 2). The fourth protein was identified as Tsetse antigen-5 (TAg-5), which is a member of a large family of anti-haemostatic proteins. The results show that these four proteins are the most abundant soluble gene products present in salivary glands of teneral G. m. morsitans. We discuss the possible functions of these major proteins in cyclical transmission of African trypanosomes.     

Transmission of MdSGHV among adult house flies, Musca domestica (Diptera: Muscidae), occurs via oral secretions and excreta

The MdSGHV is a double-stranded DNA virus that replicates in the salivary glands of infected adult house flies. Transmission of this non-occluded, enveloped virus is believed to be mediated orally via deposition and consumption of oral secretions composed of salivary gland secretions and crop contents. In this study, transmission electron micrographs of crops from infected flies showed numerous enveloped virions in the crop lumen adjacent to the cuticular intima, as well as on the hemocoel side in close vicinity to muscle cells. Oral treatments of newly emerged flies with viremic salivary gland homogenates, crop homogenates, or gradient-purified virus resulted in an average 44% infection. Virus released via oral secretion was infectious when ingested by newly emerged adult flies, resulting in an average 66% infection. Using quantitative real-time PCR, MdSGHV DNA was quantified in oral secretions and excreta obtained from viremic flies. Between 2 and 4 days post-infection (dpi), viral copy numbers in oral secretions increased exponentially and from 5 to 21 dpi each infected fly released an average 10⁶ MdSGHV copies per feeding event. Excreta samples collected overnight from individual infected flies at 5 dpi contained an average 6.5 × 10⁵ viral copies. Low but detectable infection rates were produced when newly emerged flies were challenged with excreta samples. In summary, evaluation of the quantity and infectivity of MdSGHV released by individual infected house flies clearly showed that deposition of oral secretions and excreta onto a shared food substrate is the main route of natural MdSGHV transmission among adult house flies.     

The antiviral drug valacyclovir successfully suppresses salivary gland hypertrophy virus (SGHV) in laboratory colonies of Glossina pallidipes

Many species of tsetse flies are infected with a virus that causes salivary gland hypertrophy (SGH) symptoms associated with a reduced fecundity and fertility. A high prevalence of SGH has been correlated with the collapse of two laboratory colonies of Glossina pallidipes and colony maintenance problems in a mass rearing facility in Ethiopia. Mass-production of G. pallidipes is crucial for programs of tsetse control including the sterile insect technique (SIT), and therefore requires a management strategy for this virus. Based on the homology of DNA polymerase between salivary gland hypertrophy virus and herpes viruses at the amino acid level, two antiviral drugs, valacyclovir and acyclovir, classically used against herpes viruses were selected and tested for their toxicity on tsetse flies and their impact on virus replication. While long term per os administration of acyclovir resulted in a significant reduction of productivity of the colonies, no negative effect was observed in colonies fed with valacyclovir-treated blood. Furthermore, treatment of a tsetse colony with valacyclovir for 83 weeks resulted in a significant reduction of viral loads and consequently suppression of SGH symptoms. The combination of initial selection of SGHV-negative flies by non-destructive PCR, a clean feeding system, and valacyclovir treatment resulted in a colony that was free of SGH syndromes in 33 weeks. This is the first report of the use of a drug to control a viral infection in an insect and of the demonstration that valacyclovir can be used to suppress SGH in colonies of G. pallidipes.     

Anatomy and ultrastructure of the salivary (prosomal) glands in unfed water mite larvae Piona carnea (C.L. Koch, 1836) (Acariformes: Pionidae)

Anatomy and ultrastructure of prosomal salivary glands in the unfed water mite larvae Piona carnea (C.L. Koch, 1836) were examined using serial semi-thin sections and transmission electron microscopy. Three pairs of alveolar salivary glands shown are termed lateral, ventro-lateral and medial in accordance with their spatial position. These glands belong to the podocephalic system and are situated on the common salivary duct from back to forth in the above mentioned sequence. The arrangement of the medial glands is unusual because they are situated one after another on the medial (axial) body line, therefore they are termed anterior and posterior medial glands. The secretory duct of the anterior medial gland mostly turns right, and the duct of the posterior gland turns left. The salivary glands are located in the body cavity partly inside the gnathosoma and in the idiosoma in front of the brain (synganglion). Each gland is represented by a single acinus (alveolus) and is composed of several cone shaped secretory cells arranged around the large central (intra-acinar) cavity with the secretory duct base. The cells of all glands are filled with secretory vesicles of different electron density. The remaining cell volume is occupied by elements of rough endoplasmic reticulum, and the membrane enveloping vesicles may have ribosomes on its external surface. Large nuclei provided with large nucleoli occupy the basal cell zones. The pronounced development of the prosomal salivary glands indicates their important role in extra-oral digestion of water mite larvae.     

Genome analysis of a Glossina pallidipes salivary gland hypertrophy virus reveals a novel, large, double-stranded circular DNA virus

Several species of tsetse flies can be infected by the Glossina pallidipes salivary gland hypertrophy virus (GpSGHV). Infection causes salivary gland hypertrophy and also significantly reduces the fecundity of the infected flies. To better understand the molecular basis underlying the pathogenesis of this unusual virus, we sequenced and analyzed its genome. The GpSGHV genome is a double-stranded circular DNA molecule of 190,032 bp containing 160 nonoverlapping open reading frames (ORFs), which are distributed equally on both strands with a gene density of one per 1.2 kb. It has a high A+T content of 72%. About 3% of the GpSGHV genome is composed of 15 sequence repeats, distributed throughout the genome. Although sharing the same morphological features (enveloped rod-shaped nucleocapsid) as baculoviruses, nudiviruses, and nimaviruses, analysis of its genome revealed that GpSGHV differs significantly from these viruses at the level of its genes. Sequence comparisons indicated that only 23% of GpSGHV genes displayed moderate homologies to genes from other invertebrate viruses, principally baculoviruses and entomopoxviruses. Most strikingly, the GpSGHV genome encodes homologues to the four baculoviral per os infectivity factors (p74 [pif-0], pif-1, pif-2, and pif-3). The DNA polymerase encoded by GpSGHV is of type B and appears to be phylogenetically distant from all DNA polymerases encoded by large double-stranded DNA viruses. The majority of the remaining ORFs could not be assigned by sequence comparison. Furthermore, no homologues to DNA-dependent RNA polymerase subunits were detected. Taken together, these data indicate that GpSGHV is the prototype member of a novel group of insect viruses.     

Human kallikrein 13 expression in salivary gland tumors

The human kallikrein 13 protein (hK13) is expressed in many normal tissues. Petraki et al have previously described presence of hK13 in salivary gland tissue, localized to duct epithelia and some acinar cells. The aim of this study was to determine whether hK13 is expressed in salivary gland tissues and salivary gland tumors (both benign and malignant), in order to compare normal with tumor tissues. Pleomorphic adenomas (PA), adenoid cystic carcinomas (ACC), polymorphous low grade adenocarcinomas (PLGA), acinic cell carcinomas (ACI), mucoepidermoid carcinomas (MEC) and adenocarcinomas not otherwise specified (ANOS) of both minor and major salivary glands were examined. The results of this study indicate that most salivary gland tumors show high levels of expression of hK13. Overall, staining in PA was significantly less than that seen in normal salivary gland tissue. PLGA, ACC and ANOS each stained significantly more than normal salivary gland tissue while MEC and ACI did not. Ductal cells and cells lining duct-like structures showed a higher intensity of staining than non-ductal cells in most tumors. Tumors which exhibited only non-ductal cells also exhibited cytoplasmic staining. In conclusion, we demonstrate the high expression of hK13 in several common salivary gland tumors.     

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.     

The salivary glands of the cockroach Nauphoeta cinerea (Olivier)

The innervation of the salivary gland of the cockroach Nauphoeta cinerea (Olivier) has been investigated with the use of light and scanning electron microscopy. Light microscopy of methylene blue stained glands reveals the presence of a dual innervation arising from the ventral nerve cord and the stomodeal nervous system; the principal innervation is that from the ventral nerve cord which passes to the gland via the reservoir ducts. Branches of these nerves form a plexus on the acinar surface, the axons of which exhibit swelling at irregular intervals. The presence of this surface plexus and the axonal swellings was confirmed by scanning electron microscopy both in normal glands and in those in which the basal lamina had been removed by means of an HCl-collagenase digestion method. No acinar plexus was seen to be formed by branches of the stomatogastric nerve that were associated with the gland. However, other branches of this nerve were clearly connected with a complex network of multipolar neurones on the surfaces of the anterior regions of both salivary reservoirs.     

Expression of CD44 isoforms in normal salivary gland tissue: an immunohistochemical and ultrastructural study

We studied the expression of CD44 isoforms immunoreactivity in normal human salivary gland tissue, aiming at its full characterisation in normal epithelial and myoepithelial cell types. Optical immunohistochemistry techniques using monoclonal antibodies anti-CD44v3, CD44v4/5 and, for CD44v6, together with immunoelectron microscopy, were performed in serous, seromucinous and mucinous glands. Normal human breast and a case of lactating breast adenoma were used for comparative purposes and as controls. CD44v3 was positive in acinar and myoepithelial cells and was absent in mucin-producing cells from the different gland types. CD44v4/5 was consistently negative in all types of salivary tissue. CD44v6 was constantly positive in serous acinar cells, focally positive in basal cells of ducts, and myoepithelial cells consistently expressed it. At the ultrastructural level, CD44v6 was localised to the interdigitating processes of acinar cells, whenever they were not covered by basal lamina and to the cell membrane facing myoepithelial cells. In myoepithelial cells, immunolabelling was found at the membranes facing the acinar cells and in caveolae present at this interface. No labelling was found at cell membranes of both acinar and myoepithelial cells in contact with basal lamina or at the luminal aspect of the former. The finding of CD44v3 and v6 in myoepithelium of normal salivary glands may argue in favour of the role of these molecules in the regulation of growth and renewal of normal tissues and, potentially, on the morphogenesis of salivary gland neoplasms.     

Light and electron microscopy studies of the midgut and salivary glands of second and third instars of the horse stomach bot,Gasterophilus intestinalis

A morphological study of the midgut and salivary glands of second and third instars of Gasterophilus intestinalis (De Geer) (Diptera: Oestridae) was conducted by light, scanning and transmission electron microscopy. The midgut is anteriorly delimited by a proventriculus, without caeca, and is composed of posterior foregut and anterior midgut tissue from which a double‐layered peritrophic matrix is produced. The midgut can be divided into anterior, median and posterior regions on the basis of the structural and physiological variations of the columnar cells which occur along its length. Two other types of cell were identified: regenerative cells scattered throughout the columnar cells, and, more rarely, endocrine cells of two structural types (closed and open). Different secretion mechanisms (merocrine, apocrine and microapocrine) occur along the midgut epithelium. Abundant microorganisms are observed in the endoperitrophic space of the anterior midgut. The origin and nature of these microorganisms remain unknown. No structural differences are observed between the second and third instar midguts. The salivary glands of G. intestinalis second and third instars consist of a pair of elongated tubular structures connected to efferent ducts which unite to form a single deferent duct linked dorsally to the pharynx. Several intermediate cells, without cuticle, make the junction with the salivary gland epithelium layer. Cytological characteristics of the gland epithelial cells demonstrate high cellular activity and some structural variations are noticed between the two larval stages.