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Ultrastructure of the salivary glands of non-infected and infected glands in Glossina pallidipes by the salivary glands hypertrophy virus
Journal of Invertebrate Pathology 112 (2013) S53–S61
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Ultrastructure of the salivary glands of non-infected and infected glands in Glossina pallidipes by the salivary glands hypertrophy virus Laura Guerra a,⇑, John G. Stoffolano Jr. b, Gabriella Gambellini a, Valentina Laghezza Masci a, Maria Cristina Belardinelli a, Anna Maria Fausto a a b
Dipartimento per le Innovazioni dei sistemi Biologici, Agroalimentari e Forestali, Università della Tuscia, Largo dell’Università, 01100 Viterbo, Italy Department of Plant, Soil and Insect Sciences, Division of Entomology, University of Massachusetts, Amherst, MA 01003, USA
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Article history: Available online 19 April 2012 Keywords: Hytrosavirus Tsetse fly Muscular tissue Scanning electron microscopy Transmission electron microscopy Trypanosome
a b s t r a c t Light, scanning electron, and transmission electron microscopy analyses were conducted to examine the morphology and ultrastructure of the salivary glands of Glossina pallidipes. Three distinct regions, each with a characteristic composition and organization of tissues and cells, were identified: secretory, reabsorptive and proximal. When infected with the salivary gland hypertrophy (SGH) virus, glands showed a severe hypertrophy, accompanied by profound changes in their morphology and ultrastructure. In addition, the muscular fibers surrounding the secretory region of the glands were disrupted. The morphological alterations in the muscular tissue, caused by viral infection, could be an important aspect of the pathology and may shed light on the mode of action of the SGH virus. Results were discussed with regard to the potential effect of viral infection on normal salivation and on the ability of infected tsetse flies to transmit a trypanosome parasite. Copyright Ó International Atomic Energy Agency 2013. Published by Elsevier Inc. All Rights Reserved.
1. Introduction Tsetse flies (Diptera: Glossinidae) are obligate blood feeding insects and important disease vectors transmitting different pathogenic trypanosome species that cause human sleeping sickness and livestock trypanosomiasis in Africa. Trypanosomes of the Trypanosoma brucei group, including the two human-pathogenic subspecies T. b. gambiense and T. b. rhodesiense, have to go through a complex developmental cycle in the alimentary tract and the salivary glands of the tsetse fly (Van Den Abbeele et al., 1999). Thus, the salivary glands play an important role in the transmission of the parasite. Many species of tsetse flies are infected with a virus that causes salivary gland hypertrophy (SGH). Salivary Gland Hypertrophy Viruses (SGHVs) have been found and studied not only in tsetse flies but also in house flies, Musca domestica (Lietze et al., 2011). Recently, the genomic organization of SGHVs was determined and, on the basis of the available morphological, (patho)biological, genomic, and phylogenetic data, it was proposed that these viruses are members of a new virus family, named Hytrosaviridae (AbdAlla et al., 2009). These hytrosaviruses are rod-shaped, contain large circular double-stranded DNA genomes, replicate in the nu-
⇑ Corresponding author. Fax: +39 0761357389. E-mail address: [email protected] (L. Guerra).
clei of salivary gland cells in adult flies causing distinct tissue hypertrophy, and reduce fertility of their hosts (Lietze et al., 2011). Our study focused on Glossina pallidipes, a species in which the first identification of virus particles, associated with SGH symptoms, dates to the 1970s (Jaenson, 1978). The G. pallidipes SGHV (GpSGHV), in addition to infecting the salivary glands, has been reported to replicate in the female milk gland as well as in gonadal tissues, resulting in testicular degeneration and ovarian abnormalities (Sang et al., 1999, 1998). Electron microscopic observations of virus particles, either in thin sections of hypertrophied salivary glands or from sucrose density gradient-purified, negatively stained preparations, showed enveloped bacilliform virions. The pathology of infected cells and effects on various organelles in the salivary glands have been documented (Jura et al., 1989; Kokwaro et al., 1991, 1990; Otieno et al., 1980). However, certain aspects of the interaction between the virus and host cells need further study, such as the cytopathology induced in the muscle cell layer or sheath covering the salivary glands. Therefore, the purpose of this research was to describe the overall structure of both non-infected and infected salivary glands and their morphological and ultrastructural differences, to highlight potential viral effects on the glands’ physiology, and to better understand the mode of action of SGHV in tsetse flies. Furthermore, this study may shed light on the possible relationship between the observed structural and assumed functional changes of the salivary glands and how viral infection may affect the role of tsetse flies as vectors of trypanosomes.
0022-2011/$ - see front matter Copyright Ó International Atomic Energy Agency 2013. Published by Elsevier Inc. All Rights Reserved. http://dx.doi.org/10.1016/j.jip.2012.04.003
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2. Materials and methods 2.1. Animals Pupae of G. pallidipes were received from a laboratory colony maintained in the Insect Pest Control Laboratory of the International Atomic Energy Agency (IAEA) in Vienna, Austria. These pupae were maintained at 24 °C, 70% relative humidity and a photoperiod of 12 h as described in previous studies (Abd-Alla et al., 2007; Feldmann, 1994; Gooding et al., 1997). Adults were fed with sugar solution and their average survival was 10 days. Specimens of both sexes of the adult insects were dissected to obtain samples of healthy salivary glands and virus-infected hypertrophied glands. 2.2. Light microscopy Samples of salivary glands, infected and non-infected, were dissected in PBS and immediately observed using a computerized image analysis system, which included a Zeiss light microscope (Axiophot) equipped with a video color camera (Axio Cam MRC, Arese, Milano-Italy) and imaging software (KS 300 and AxioVision). 2.3. Scanning electron microscopy (SEM) For the SEM investigation, infected and non-infected salivary gland samples were fixed for one night in 2.5% glutaraldehyde in 0.1 M cacodylate 3% sucrose buffer pH 7.2. Subsequently, the material was washed in 0.1 M cacodylate buffer overnight and then post-fixed with 1% osmium tetroxide in 0.1 M cacodylate buffer. The specimens were dehydrated through a series of steps in acetone at progressively increasing concentrations (from 50% to 100%). The dehydrated material was dried with liquid carbon dioxide using a critical point dryer (Balzer Union CPD 020), mounted on a special sample holder, and coated using a gold evaporator (Balzer Union MD 010). So treated, the samples were observed under a Jeol JSM 5200 microscope. 2.4. Transmission electron microscopy (TEM) For the TEM survey, samples of infected and non-infected salivary glands were fixed and dehydrated as described for the SEM analysis and then infiltrated in Epon resin (TAAB, England). Thin sections were cut with Reichert Ultracut and LKB Nova ultramicrotomes. Semithin sections (1 lm thick) collected on slides were stained with toluidine blue and then observed with Zeiss Axiophot light microscopy. Ultra-thin (thickness of 60–80 nm) sections were collected on copper grids, stained with uranyl acetate and lead citrate and observed at 120 kV using a Jeol JEM EX II transmission electron microscope, equipped by a Veleta CCD camera (Olympus) and the pictures where processed by iTEM software. 3. Results The salivary glands of G. pallidipes consisted of two tubular, thin and transparent structures that extended into the abdomen on either side of the gut. In the normal and healthy salivary glands, each paired gland showed three distinct regions: secretory, reabsorptive, and proximal. This last portion continued in a duct joining a common salivary duct (Fig. 1A). The external morphology of healthy salivary glands was investigated by light microscopy and SEM. The distal, secretory region extended from the abdomen into the thorax and was surrounded by a thick muscle tissue with evident longitudinal muscle fibers (Fig. 1D and E), whereas the outer
surfaces of the reabsorptive and proximal regions were devoid of a muscular coat (Figs. 1A–C). The infection with SGH virus greatly enlarged the salivary glands, and this hypertrophy was uniform over the entire length of the gland. In fact, each individual salivary gland was affected equally, increased in diameter, and appeared whitish and pale, thus being easily distinguishable from non-infected and transparent glands (Fig. 2A). However, hypertrophy primarily affected the secretory region of the glands, which filled most of the abdominal cavity and became entangled with fat body and tracheae (Fig. 2A). Scanning electron micrographs revealed an evident alteration of the muscular tissue in the hypertrophied salivary glands; the longitudinal muscle fibers lost their organization and arrangement and, in several places, were disrupted (Fig. 2D and E). Figs. 3 and 4 show and compare transmission electron micrographs obtained from the secretory regions of normal and hypertrophied salivary glands, respectively. Both light microscopy and TEM examination revealed that the secretory region of the normal gland was characterized by a muscular coat surrounding the gland, an epithelium consisting of a single layer of cells, and a central lumen (Fig. 3A and B). TEM observations of cross sections of this region showed that the external muscle tissue was composed of large muscle cells, each showing numerous myofibrils (Fig. 3C). The secretory cells were separated from the muscle by a basement membrane and contained an extensive rough endoplasmic reticulum, many Golgi complexes, numerous mitochondria, some of which had electron-dense granules, and a large number of secretory granules (Fig. 3D, F and G). Especially in the apical area of the epithelium, adjacent cells were in contact along their lateral plasma membranes by septate desmosomes (Fig. 3E). In addition, the secretory cells showed microvilli extending into the gland lumen and involved in the release of secretion (Figs. 3E and H). The lumen contained an electron-dense matrix with numerous electron-opaque filaments (Fig. 3E). In the secretory region of the hypertrophied glands, infected by SGH virus, the muscular coat surrounding the gland underwent profound ultrastructural changes (Fig. 4A–C). Transmission electron micrographs showed the presence of vacuoles in the muscle cells, even in the region contacting the underlying basement membrane (Fig. 4C). Owing to the remarkable degree of hypertrophy, this basement membrane had an irregular and not well defined structure, appeared stratified and thicker than the same region of the healthy salivary glands, and showed evident changes in the relationship with the muscle cells (Fig. 4C). Moreover, cells of the glandular epithelium were highly vacuolated (Fig. 4A and B), cell junctions were no longer visible, and the gland enlargement was caused by cellular proliferation of the secretory cells (Fig. 4D), resulting in an abnormal multilayered epithelium and a reduced gland lumen (Fig. 4A). In certain areas, the proliferating cells lost contact with the degenerated basement membrane because of the vacuolization of the cytoplasm (Fig. 4C). Numerous virus particles were scattered in the nuclei and in the cytoplasm of secretory cells (Fig. 4E and F). Fig. 4E depicts the morphological differences of the virus in the nucleus and in the cytoplasm; in the nucleus, the virus replicated and assembled its nucleocapsid, and with the transition into the cytoplasm, the viral particles further developed. The high-magnification micrograph in Fig. 4G shows longitudinal and cross sections of the SGH virus particles in the cytoplasm of the secretory cells. In the normal salivary glands, with the transition from the secretory to the reabsorptive and then to the proximal region, the diameter of the gland decreased (Fig. 2A). The reabsorptive region was localized in the thorax of the insect, and at the electron microscope level this region was characterized by an external basal lamina with a smooth outside surface and underlying epithelial
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Fig. 1. Light and scanning electron microscopy (SEM) of the general organization of healthy salivary glands in G. pallidipes. (A) The salivary glands are characterized by three distinct regions: secretory, reabsorptive, and proximal. Bar = 100 lm. (B nad C) Light microscopy magnifications of the proximal and reabsorptive regions. Bars = 20 lm. (D and E) SEM of the secretory region of the salivary glands. Micrographs show the longitudinal muscle fibers surrounding this region of the salivary glands. Bars = 50 lm and 10 lm, respectively.
cells showing an infolded basal membrane (Fig. 5A and B). This region was very rich in microvilli that extended from the epithelium into the lumen, and the cells were involved in the reabsorption of the lumen secretion (Fig. 5A and B). Furthermore, the secretion in the lumen of this gland region contained fewer electron-dense filaments than that of the secretory region (Fig. 5A). Finally, the morphology of the proximal region was characterized by a thin basement membrane that surrounded an epithelial layer. The epithelial cells were separated from the lumen by a cuticle consisting of a light layer (endocuticle) and a dense, dark, thin layer (epicuticle) (Fig. 5C). Infection with SGH virus also affected the reabsorptive region, where the ultrastructural analysis showed a profound change of the internal organization of tissues. The epithelium appeared stratified and vacuolated, and it was difficult to distinguish the bound-
ary between epithelium and lumen (Fig. 6A). The reabsorptive cells had enormous cytoplasmic vacuoles and the microvilli appeared destroyed or damaged (Fig. 6A). In the reduced glandular lumen, it was possible to identify viral particles (Fig. 6B). The basement membrane was thicker and less compact than that of the same region of healthy salivary glands (Fig. 6C). Moreover, in the reabsorptive cells, free ribosomes, scattered strands of rough endoplasmic reticulum, and numerous degenerated mitochondria were present (Fig. 6C and D). The viral infection also affected the proximal region (Fig. 2A), but duct epithelial cells of the salivary glands underwent minor changes compared with the secretory and reabsorptive cells (Fig. 6E). No viral particles were observed in this gland region. In addition, TEM observations revealed the absence of secretion in the lumen (Fig. 6E).
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Fig. 2. Light and scanning electron microscopy (SEM) of the hypertrophied salivary glands, infected by SGH virus, in G. pallidipes. (A) The hypertrophy involves the entire gland, especially the secretory region, which is entangled with tracheae in the abdomen of the fly. Bar = 200 lm. (B and C) Light microscopy magnifications of the junction between the two glands and the transition between the proximal and reabsorptive regions. Bars = 20 lm. (D and E) SEM of the secretory region of the salivary glands. The muscle tissue undergoes a profound alteration (i.e., muscle fibers lose their organization and often are interrupted or destroyed producing holes in the sheath). Bars = 100 lm and 25 lm, respectively.
4. Discussion Based on their anatomy, insect salivary glands can be divided into two groups (House and Ginsborg, 1985). Salivary glands of the higher Diptera consist of simple tubules joined to form a common duct, whereas in other insect orders the salivary glands are more complex, having a racemose appearance with ducts and acini. Several ultrastructural studies of dipteran salivary glands have been published (House and Ginsborg, 1985; Janzen and Wright, 1971; Martoja and Ballan-Dufrançais, 1984; Wright, 1969).
In this study, a detailed reconstruction of the general organization of the salivary glands of G. pallidipes was obtained by both SEM and TEM analyses. Along the length of this organ, we could distinguish three regions, which differed in their morphology and ultrastructure: a secretory, a reabsorptive, and a proximal region. A similar division of the salivary glands was observed in other insects, as in Calliphora, where the ultrastructural and physiological observations showed a secretory distal region and a proximal reabsorptive tract (Oschman and Berridge, 1970).
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Fig. 3. Light and transmission electron micrographs of the secretory region in the normal, uninfected salivary glands. (A and B) Light and transmission electron micrographs, respectively, showing the muscular sheath that surround the glands, an epithelium composed of a single layer of cells, and a central lumen. Bars = 10 lm and 1 lm, respectively. (C) The muscular sheath shows the muscle fibers and it is separated from the epithelium by the basement membrane. Bar = 250 nm. (D) Magnification of a secretory cell showing the rough endoplasmic reticulum. Bar = 200 nm. (E) This transmission electron micrograph of the secretory cell depicts the presence of microvilli that extend into the lumen, a septate desmosome () in the apical cell membrane, and a nucleus. The lumen contains an electron-dense matrix, where there are numerous electron-opaque filaments surrounded by clear zones. Bars = 500 nm, () 50 nm. (F and G) The secretory cell contains Golgi complexes, mitochondria, and secretory granules. Bars = 50 nm and 100 nm, respectively. (H) Magnification of microvilli involved in the luminal secretion and cross sections of the electron-opaque filaments surrounded by clear zones. Bar = 100 nm. Bm, basement membrane; Ep, epithelium; Gc, Golgi complexes; Lu, lumen; M, mitochondria; Mc, muscular coat; Mv, microvilli; N, nucleus; Rer, rough endoplasmic reticulum; Sg, secretory granules.
SEM and TEM investigation of the secretory region of the G. pallidipes salivary glands allowed an accurate description of the morphology and ultrastructure of a thick muscle tissue sheath, composed of longitudinal muscular fibers that covered this region. This muscle layer may be responsible for the contraction necessary to expel the saliva, as described in other insects (Reis et al., 2003). The presence of external muscle tissue in the salivary glands varies among insects. In fact, salivary glands of Anopheles stephensi (Wright, 1969), Aedes aegypti (Janzen and Wright, 1971), and of Calliphora (Oschman and Berridge, 1970) show no muscle covering. The same is true for the salivary glands of adult Ceratitis capitata, and it has been suggested that the actin filament network may generate peristaltic contractions that move the saliva along the lumen (Riparbelli et al., 1994). Nevertheless, in the salivary glands of
other insects muscle tissue has been observed, as in Cimex hemipterus (Serrão et al., 2008) and in Triatoma infestans (Reis et al., 2003). In previous studies on tsetse flies, very little attention was paid to the involvement of the muscle sheath in the secretion process and how the secretion is discharged from the extracellular cavity. No study has focused on the normal secretory process of the salivary glands of tsetse flies even though basic information about salivary gland secretion is available from a model dipteran (Berridge and Patel, 1968; Hansen Bay, 1978; House and Ginsborg, 1985). Van Den Abbeele et al. (2010, 2007) examined the molecular aspects of the sialome of tsetse to determine the constituents of saliva, especially in relation to possible changes in the composition of the secretion caused by infection with trypanosomes, but the overall physiology appears to have been ignored.
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Fig. 4. Light and transmission electron micrographs of the secretory region in the hypertrophied salivary glands, infected by SGH virus. (A and B) Light and transmission electron micrographs, respectively, showing the alteration of the muscular sheath, the vacuolization of the epithelium, and the significantly reduced lumen. Bars = 20 lm and 2 lm, respectively. (C) In the muscle, the arrangement of muscle fibers undergoes a profound alteration and the basement membrane is degenerated. Bar = 1 lm. (D) This magnification of the epithelium shows the cell proliferation induced by the viral infection. Bar = 5 lm. (E and F) The viral particles replicate and form their nucleocapsids in the nucleus and then undergo a further development in the cytoplasm of the secretory cells. Bars = 200 nm and 100 nm, respectively. (G) High magnification of the viral particles in the cytoplasm. Bar = 100 nm. Bm, basement membrane; Ep, epithelium; Lu, lumen; Mc, muscular coat; N, nucleus; Vp, viral particles.
The secretory cells of the salivary glands of G. pallidipes contained a high number of large mitochondria that had electrondense granules, characteristic of secretory cells. These cell types possessed an extensive rough endoplasmic reticulum, Golgi complexes and numerous secretory granules, suggesting an important role of these cells in the secretion of enzymes, ions and water, as described in previous studies (House and Ginsborg, 1985; Kokwaro et al., 1991). Studies performed on Calliphora suggest that the secretory granules contain amylase, the only enzyme that has been found in the saliva of this insect. These secretion granules are probably formed in the Golgi complex, a process that has been documented clearly in other studies (Oschman and Berridge, 1970). The reabsorptive region of the G. pallidipes salivary glands was devoid of a muscular layer and the basement membrane was in direct contact with hemolymph, similar to the reabsorptive portion of the salivary glands of Calliphora (Oschman and Berridge,
1970). The basal domain of the plasma membrane of the epithelial cells was infolded, a large number of microvilli extended from the epithelium into the lumen, and the secretion in the lumen appeared more dilute than that of the secretory region, suggesting an active reabsorption in this region and a change in the composition of the saliva. Morphological and physiological studies performed on Calliphora support this hypothesis; epithelial cells of this region are involved in the reabsorption of ions, transforming the luminal fluid into a hypo-osmotic final saliva (Oschman and Berridge, 1970; Rotte et al., 2008). The proximal duct of the salivary glands was poorly investigated in tsetse flies (Kokwaro et al., 1991) and in other insects. Our results demonstrated that epithelial cells produced a cuticle whose morphology resembled that described in the crop of Phormia regina, an important organ of the alimentary tract of insects (Stoffolano et al., 2010) and indicative of foregut organization.
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Fig. 5. Transmission electron micrographs of the reabsorptive and proximal regions of the healthy salivary glands. (A and B) The reabsorptive region is bordered by a basement membrane, which is in direct contact with the hemolymph. This region shows a large number of microvilli that extend from the epithelium into the lumen and are involved in the reabsorption of the luminal secretion. Bars = 1 lm and 500 nm, respectively. (C) The proximal region is characterized by a thin basement membrane and an epithelial layer. The epithelial cells are separated from the lumen by a cuticle that consists of an internal endocuticle and an external epicuticle. Bar = 1 lm. Bm, basement membrane; Enc, endocuticle; Ep, epithelium; Epc, epicuticle; Lu, lumen; Mv, microvilli.
Important results of this study were obtained from the analysis of the profound morphological and ultrastructural changes of the G. pallidipes salivary glands, caused by the SGH virus. The viral infection produced a significant hypertrophy throughout the entire gland, but the highest degree of this hypertrophy affected the secretory region. Our SEM and TEM observations of this region showed severe alterations of the muscular coat and muscle cells, although viral particles were not detected in these cells. The
Fig. 6. Transmission electron micrographs of the reabsorptive and proximal regions in hypertrophied salivary glands. (A) The reabsorptive region shows a degenerated ultrastructure; the epithelium is vacuolated and the microvilli appear destroyed. Bar = 2 lm. (B) In the reduced gland lumen few viral particles are present. Bar = 250 nm. (C) The basement membrane is thicker and more stratified than the same region of the healthy salivary glands. Bar = 200 nm. (D) The reabsorptive cells are altered and show degenerated mitochondria. The arrow indicates scattered strands of rough endoplasmic reticulum. Bar = 100 nm. (E) A lesser degree of ultrastructural alteration seems to affect the proximal region of the salivary glands, where no viral particles were observed. Bar = 500 nm. Bm, basement membrane; Enc, endocuticle; Ep, epithelium; Epc, epicuticle; Lu, lumen; M, mitochondria; Vp, viral particles.
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longitudinal muscle fibers lost their organization, suggesting a degeneration of the myofibrils, which in normal muscle structure remain intact for proper function. In addition, the underlying basement membrane was irregular, appeared striated, and the changes of its relationships with muscle cells and proliferating epithelial cells suggest a degeneration of cytoskeletal elements that affect communication of the basement membrane with the surrounding cells. Based on these results, we can hypothesize that the contractile capacity of the muscle sheath that covers the gland is greatly compromised, which should lead to a decreased ability to expel saliva. This hypothesis is supported further by the reduction of the volume of the glandular secretion in the infected salivary glands. The disease pathology also affected the reabsorptive region of the salivary glands, where the epithelial cells were rich in degenerated mitochondria and free ribosomes, and several strands showed interruption of the rough endoplasmic reticulum, denoting a possible altered cellular activity. Moreover, our ultrastructural observations showed the lack of microvilli in this region, suggesting a compromised reabsorptive function of the infected salivary glands. Thus, although it is not known whether the enzyme composition and/or functions of hypertrophied salivary glands are affected negatively by the disease pathology, the results obtained in this study highlight the morphological and ultrastructural changes of the whole salivary gland, which are viral-induced and could be related to an overall alteration of gland function. These comments are essential to correlate the action of the SGH virus to fly infection with Trypanosoma spp. In the normal parasite infection, trypanosome development takes place in the salivary glands of tsetse flies. In particular, four sequential stages of development can be recognized in the gland: (1) the uncoated epimastigote trypanosomes (with a prenuclear kinetoplast) attached by elaborate flagellar outgrowths that ramify between host microvilli; (2) uncoated premetacyclic trypomastigotes (with postnuclear kinetoplast) and reduced flagellar outgrowths; (3) nascent metacyclic trypomastigotes that have acquired the surface coat and lost their flagellar outgrowths but remain attached to microvilli; (4) unattached coated metacyclics lying free in the saliva ready for discharge when the fly bites (Tetley and Vickerman, 1985). Previous studies reported that the impact of SGHV infection on trypanosome transmission is unclear (Burtt, 1945; Otieno et al., 1980; Whitnall, 1934). Kokwaro et al. (1991) examined the mixed infection of tsetse salivary glands, evidencing a severe cellular disintegration and a significant degeneration of trypanosomes in these cells. Based on the results obtained in this study, we can hypothesize that the ultrastructural alterations of the glandular epithelium, caused by viral infection, could impair the integrity of the structures necessary to the development and survival of trypanosomes in the salivary glands. Therefore, this observation may in part explain the degeneration of trypanosomes, as described above, and we can assume that the SGH virus could affect the normal ability of viral infected tsetse flies to transmit trypanosomes. Nevertheless, further studies are necessary to elucidate the mode of action of this virus, especially in relation to the possible physiological changes of the salivary glands, to better understand how to best manage the SGH virus in tsetse flies, which are important vectors of trypanosomes, and to explore alternative ways to eliminate the virus from the colonies being maintained for sterile insect control using massive release programs.
Disclosures Each author of this paper have not any involvement, financial or otherwise, that might potentially bias their work.
Acknowledgments We gratefully acknowledge the financial support for this study provided by the research Contract No. 16419 with International Atomic Energy Agency (IAEA). References Abd-Alla, A., Vlak, J.M., Bergoin, M., Maruniak, J.E., Parker, A., Burand, J., Jehle, J.A., Boucias, D., 2009. Hytrosaviridae: a proposal for classification and nomenclature of a new insect virus family. Arch. Virol. 154, 909–918. Abd-Alla, A., Bossin, H., Cousserans, F., Parker, A., Bergoin, M., Robinson, A., 2007. Development of a non-destructive PCR method for detection of the salivary gland hypertrophy virus (SGHV) in tsetse flies. J. Virol. Method 139, 143–149. Berridge, M.J., Patel, N.G., 1968. Insect salivary glands: stimulation of fluid secretion by 5-hydroxytryptamine and adenosine-30 , 50 -monophosphate. Science 162, 462–463. Burtt, E., 1945. Hypertrophied salivary glands in Glossina: evidence that G. pallidipes with this abnormality is peculiarly to trypanosome infection. Ann. Trop. Med. Parasitol. 39, 11–13. Feldmann, U., 1994. Guidelines for the rearing of tsetse flies using the membrane feeding technique. in: Ochieng’-Odero, J.P.R. (Ed.), Techniques of Insect Rearing for the Development of Integrated Pest and Vector Management Strategies. ICIPE Science Press, Nairobi, Kenya, pp. 449–471. Gooding, R.H., Feldmann, U., Robinson, A.S., 1997. Care and maintenance of tsetse colonies. In: Crampton, J.M., Beard, C.B., Louis, C. (Eds.), The Molecular Biology of Insect Disease Vectors: A Methods Manual. Chapman & Hall Ltd., London, UK, pp. 41–55. Hansen Bay, C.M., 1978. Control of salivation in the blowfly Calliphora. J. Exp. Biol. 75, 189–201. House, C.R., Ginsborg, B.L., 1985. Salivary gland. in: Kerkut, G.A., Gilbert, L.I. (Eds.), Comprehensive Insect Physiology, Biochemistry and Pharmacology, vol. 11. Pergamon Press, New York, pp. 195–224. Jaenson, T.G.T., 1978. Virus-like rods associated with salivary gland hyperplasia in tsetse, Glossina pallidipes. Trans. Roy. Soc. Trop. Med. Hyg. 72, 234–238. Janzen, H.G., Wright, K.A., 1971. The salivary glands of Aedes aegypti (L.): an electron microscope study. Can. J. Zool. 49, 1343–1345. Jura, W.G.Z.O., Otieno, L.H., Chimtawi, M., 1989. Ultrastructural evidence for transovum transmission of the DNA virus of tsetse Glossina pallidipes (Diptera: Glossinidae). Curr. Microbiol. 18, 1–4. Kokwaro, E.D., Otieno, L.H., Chimtawi, M., 1991. Salivary glands of the tsetse Glossina pallidipes Austen infected with Trypanosoma brucei and virus particles: ultrastructural study. Insect Sci. Appl. 12, 661–669. Kokwaro, E.D., Nyindo, M., Chimtawi, M., 1990. Ultrastructural changes in salivary glands of tsetse, Glossina morsitans morsitans, infected with virus and rickettsialike organisms. J. Invertebr. Pathol. 56, 337–346. Lietze, V.U., Abd-Alla, A.M.M., Vreysen, M.J.B., Geden, C.J., Boucias, D.G., 2011. Salivary gland hypertrophy viruses: a novel group of insect pathogenic viruses. Ann. Rev. Entomol. 56, 63–80. Martoja, R., Ballan-Dufrançais, C., 1984. The ultrastruscture of the digestive and excretory organs. in: King, R.C., Akai, H. (Eds.), Insect ultrastructure, vol. 2. Plenum Press, New York, pp. 199–261. Oschman, J.L., Berridge, M.J., 1970. Structural and functional aspects of salivary fluid secretion in Calliphora. Tissue Cell 2 (2), 281–310. Otieno, L.H., Kokwaro, E.D., Chimtawi, M., Onyango, P., 1980. Prevalence of enlarged salivary glands in wild populations of Glossina pallidipes in Kenya, with a note on the ultrastructure of the affected organ. J. Invertebr. Pathol. 36, 113–118. Reis, M.M., Meirelles, M.S., Soares, M.J., 2003. Fine structure of the salivary glands of Triatoma infestans (Hemiptera: Reduviidae). Tissue Cell 35, 393–400. Riparbelli, M.G., Callaini, G., Dallai, R., 1994. Cytoskeleton of larval and adult salivary glands of the dipteran Ceratitis capitata. Implication of microfilaments and microtubules in saliva discharge. Boll. Zool. 61, 9–17. Rotte, C., Walz, B., Baumann, O., 2008. Morphological and functional characterization of the thoracic portion of blowfly salivary glands. Arthropod Struct. Develop. 37, 372–382. Sang, R.C., Jura, W.G., Otieno, L.H., Mwangi, R.W., Ogaja, P., 1999. The effects of a tsetse DNA virus infection on the functions of the male accessory reproductive gland in the host fly Glossina morsitans centralis (Diptera; Glossinidae). Curr. Microbiol. 38, 349–354. Sang, R.C., Jura, W.G., Otieno, L.H., Mwangi, R.W., 1998. The effects of a DNA virus infection on the reproductive potential of female tsetse flies, Glossina morsitans centralis and Glossina morsitans morsitans (Diptera: Glossinidae). Mem. Inst. Oswaldo Cruz. 93, 861–864. Serrão, J.E., Castrillon, M.I., Santos-Mallet, J.R., Zanuncio, J.C., Gonçalves, T.C., 2008. Ultrastructure of the salivary glands in Cimex hemipterus (Hemiptera: Cimicidae). J. Med. Entomol. 45 (6), 991–999. Stoffolano Jr., J.G., Guerra, L., Carcupino, M., Gambellini, G., Fausto, A.M., 2010. The diverticulated crop of adult Phormia regina. Arthropod Struct. Develop. 39 (4), 251–260. Tetley, L., Vickerman, K., 1985. Differentiation in Trypanosoma brucei: host-parasite cell junctions and their persistence during acquisition of the variable antigen coat. J. Cell Sci. 74, 1–19. Van Den Abbeele, J., Caljon, G., De Ridder, K., De Baetselier, P., Coosemans, M., 2010. Trypanosoma brucei modifies the tsetse salivary composition, altering the fly
L. Guerra et al. / Journal of Invertebrate Pathology 112 (2013) S53–S61 feeding behavior that favors parasite transmission. PLoS Pathog. 6 (6), e1000926. Van Den Abbeele, J., Caljon, G., Dierick, J.F., Moens, L., De Ridder, K., Coosemans, M., 2007. The Glossina morsitans tsetse fly saliva: general characteristics and identification of novel salivary proteins. Insect Biochem. Molec. 37, 1075–1085.
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Van Den Abbeele, J., Claes, Y., Van Bockstaele, D., Le Ray, D., Coosemans, M., 1999. Trypanosoma brucei spp. Development in the tsetse fly: characterization of the post-mesocyclic stages in the foregut and proboscis. Parasitol 118, 469–478. Whitnall, A.B.M., 1934. The trypanosome infections of Glossina pallidipes in the Umfolosi game reserve, Zuhuland. Onderstepoort J. Vet. Sci. Anim. Ind. 11, 7–21. Wright, K.A., 1969. The anatomy of salivary glands of Anopheles stephensi liston. Can. J. Zool. 47, 579–587.
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Ultrastructure of the salivary glands of non-infected and infected glands in Glossina pallidipes by the salivary glands hypertrophy virus Laura Guerra a,⇑, John G. Stoffolano Jr. b, Gabriella Gambellini a, Valentina Laghezza Masci a, Maria Cristina Belardinelli a, Anna Maria Fausto a a b
Dipartimento per le Innovazioni dei sistemi Biologici, Agroalimentari e Forestali, Università della Tuscia, Largo dell’Università, 01100 Viterbo, Italy Department of Plant, Soil and Insect Sciences, Division of Entomology, University of Massachusetts, Amherst, MA 01003, USA
a r t i c l e
i n f o
Article history: Available online 19 April 2012 Keywords: Hytrosavirus Tsetse fly Muscular tissue Scanning electron microscopy Transmission electron microscopy Trypanosome
a b s t r a c t Light, scanning electron, and transmission electron microscopy analyses were conducted to examine the morphology and ultrastructure of the salivary glands of Glossina pallidipes. Three distinct regions, each with a characteristic composition and organization of tissues and cells, were identified: secretory, reabsorptive and proximal. When infected with the salivary gland hypertrophy (SGH) virus, glands showed a severe hypertrophy, accompanied by profound changes in their morphology and ultrastructure. In addition, the muscular fibers surrounding the secretory region of the glands were disrupted. The morphological alterations in the muscular tissue, caused by viral infection, could be an important aspect of the pathology and may shed light on the mode of action of the SGH virus. Results were discussed with regard to the potential effect of viral infection on normal salivation and on the ability of infected tsetse flies to transmit a trypanosome parasite. Copyright Ó International Atomic Energy Agency 2013. Published by Elsevier Inc. All Rights Reserved.
1. Introduction Tsetse flies (Diptera: Glossinidae) are obligate blood feeding insects and important disease vectors transmitting different pathogenic trypanosome species that cause human sleeping sickness and livestock trypanosomiasis in Africa. Trypanosomes of the Trypanosoma brucei group, including the two human-pathogenic subspecies T. b. gambiense and T. b. rhodesiense, have to go through a complex developmental cycle in the alimentary tract and the salivary glands of the tsetse fly (Van Den Abbeele et al., 1999). Thus, the salivary glands play an important role in the transmission of the parasite. Many species of tsetse flies are infected with a virus that causes salivary gland hypertrophy (SGH). Salivary Gland Hypertrophy Viruses (SGHVs) have been found and studied not only in tsetse flies but also in house flies, Musca domestica (Lietze et al., 2011). Recently, the genomic organization of SGHVs was determined and, on the basis of the available morphological, (patho)biological, genomic, and phylogenetic data, it was proposed that these viruses are members of a new virus family, named Hytrosaviridae (AbdAlla et al., 2009). These hytrosaviruses are rod-shaped, contain large circular double-stranded DNA genomes, replicate in the nu-
⇑ Corresponding author. Fax: +39 0761357389. E-mail address: [email protected] (L. Guerra).
clei of salivary gland cells in adult flies causing distinct tissue hypertrophy, and reduce fertility of their hosts (Lietze et al., 2011). Our study focused on Glossina pallidipes, a species in which the first identification of virus particles, associated with SGH symptoms, dates to the 1970s (Jaenson, 1978). The G. pallidipes SGHV (GpSGHV), in addition to infecting the salivary glands, has been reported to replicate in the female milk gland as well as in gonadal tissues, resulting in testicular degeneration and ovarian abnormalities (Sang et al., 1999, 1998). Electron microscopic observations of virus particles, either in thin sections of hypertrophied salivary glands or from sucrose density gradient-purified, negatively stained preparations, showed enveloped bacilliform virions. The pathology of infected cells and effects on various organelles in the salivary glands have been documented (Jura et al., 1989; Kokwaro et al., 1991, 1990; Otieno et al., 1980). However, certain aspects of the interaction between the virus and host cells need further study, such as the cytopathology induced in the muscle cell layer or sheath covering the salivary glands. Therefore, the purpose of this research was to describe the overall structure of both non-infected and infected salivary glands and their morphological and ultrastructural differences, to highlight potential viral effects on the glands’ physiology, and to better understand the mode of action of SGHV in tsetse flies. Furthermore, this study may shed light on the possible relationship between the observed structural and assumed functional changes of the salivary glands and how viral infection may affect the role of tsetse flies as vectors of trypanosomes.
0022-2011/$ - see front matter Copyright Ó International Atomic Energy Agency 2013. Published by Elsevier Inc. All Rights Reserved. http://dx.doi.org/10.1016/j.jip.2012.04.003
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2. Materials and methods 2.1. Animals Pupae of G. pallidipes were received from a laboratory colony maintained in the Insect Pest Control Laboratory of the International Atomic Energy Agency (IAEA) in Vienna, Austria. These pupae were maintained at 24 °C, 70% relative humidity and a photoperiod of 12 h as described in previous studies (Abd-Alla et al., 2007; Feldmann, 1994; Gooding et al., 1997). Adults were fed with sugar solution and their average survival was 10 days. Specimens of both sexes of the adult insects were dissected to obtain samples of healthy salivary glands and virus-infected hypertrophied glands. 2.2. Light microscopy Samples of salivary glands, infected and non-infected, were dissected in PBS and immediately observed using a computerized image analysis system, which included a Zeiss light microscope (Axiophot) equipped with a video color camera (Axio Cam MRC, Arese, Milano-Italy) and imaging software (KS 300 and AxioVision). 2.3. Scanning electron microscopy (SEM) For the SEM investigation, infected and non-infected salivary gland samples were fixed for one night in 2.5% glutaraldehyde in 0.1 M cacodylate 3% sucrose buffer pH 7.2. Subsequently, the material was washed in 0.1 M cacodylate buffer overnight and then post-fixed with 1% osmium tetroxide in 0.1 M cacodylate buffer. The specimens were dehydrated through a series of steps in acetone at progressively increasing concentrations (from 50% to 100%). The dehydrated material was dried with liquid carbon dioxide using a critical point dryer (Balzer Union CPD 020), mounted on a special sample holder, and coated using a gold evaporator (Balzer Union MD 010). So treated, the samples were observed under a Jeol JSM 5200 microscope. 2.4. Transmission electron microscopy (TEM) For the TEM survey, samples of infected and non-infected salivary glands were fixed and dehydrated as described for the SEM analysis and then infiltrated in Epon resin (TAAB, England). Thin sections were cut with Reichert Ultracut and LKB Nova ultramicrotomes. Semithin sections (1 lm thick) collected on slides were stained with toluidine blue and then observed with Zeiss Axiophot light microscopy. Ultra-thin (thickness of 60–80 nm) sections were collected on copper grids, stained with uranyl acetate and lead citrate and observed at 120 kV using a Jeol JEM EX II transmission electron microscope, equipped by a Veleta CCD camera (Olympus) and the pictures where processed by iTEM software. 3. Results The salivary glands of G. pallidipes consisted of two tubular, thin and transparent structures that extended into the abdomen on either side of the gut. In the normal and healthy salivary glands, each paired gland showed three distinct regions: secretory, reabsorptive, and proximal. This last portion continued in a duct joining a common salivary duct (Fig. 1A). The external morphology of healthy salivary glands was investigated by light microscopy and SEM. The distal, secretory region extended from the abdomen into the thorax and was surrounded by a thick muscle tissue with evident longitudinal muscle fibers (Fig. 1D and E), whereas the outer
surfaces of the reabsorptive and proximal regions were devoid of a muscular coat (Figs. 1A–C). The infection with SGH virus greatly enlarged the salivary glands, and this hypertrophy was uniform over the entire length of the gland. In fact, each individual salivary gland was affected equally, increased in diameter, and appeared whitish and pale, thus being easily distinguishable from non-infected and transparent glands (Fig. 2A). However, hypertrophy primarily affected the secretory region of the glands, which filled most of the abdominal cavity and became entangled with fat body and tracheae (Fig. 2A). Scanning electron micrographs revealed an evident alteration of the muscular tissue in the hypertrophied salivary glands; the longitudinal muscle fibers lost their organization and arrangement and, in several places, were disrupted (Fig. 2D and E). Figs. 3 and 4 show and compare transmission electron micrographs obtained from the secretory regions of normal and hypertrophied salivary glands, respectively. Both light microscopy and TEM examination revealed that the secretory region of the normal gland was characterized by a muscular coat surrounding the gland, an epithelium consisting of a single layer of cells, and a central lumen (Fig. 3A and B). TEM observations of cross sections of this region showed that the external muscle tissue was composed of large muscle cells, each showing numerous myofibrils (Fig. 3C). The secretory cells were separated from the muscle by a basement membrane and contained an extensive rough endoplasmic reticulum, many Golgi complexes, numerous mitochondria, some of which had electron-dense granules, and a large number of secretory granules (Fig. 3D, F and G). Especially in the apical area of the epithelium, adjacent cells were in contact along their lateral plasma membranes by septate desmosomes (Fig. 3E). In addition, the secretory cells showed microvilli extending into the gland lumen and involved in the release of secretion (Figs. 3E and H). The lumen contained an electron-dense matrix with numerous electron-opaque filaments (Fig. 3E). In the secretory region of the hypertrophied glands, infected by SGH virus, the muscular coat surrounding the gland underwent profound ultrastructural changes (Fig. 4A–C). Transmission electron micrographs showed the presence of vacuoles in the muscle cells, even in the region contacting the underlying basement membrane (Fig. 4C). Owing to the remarkable degree of hypertrophy, this basement membrane had an irregular and not well defined structure, appeared stratified and thicker than the same region of the healthy salivary glands, and showed evident changes in the relationship with the muscle cells (Fig. 4C). Moreover, cells of the glandular epithelium were highly vacuolated (Fig. 4A and B), cell junctions were no longer visible, and the gland enlargement was caused by cellular proliferation of the secretory cells (Fig. 4D), resulting in an abnormal multilayered epithelium and a reduced gland lumen (Fig. 4A). In certain areas, the proliferating cells lost contact with the degenerated basement membrane because of the vacuolization of the cytoplasm (Fig. 4C). Numerous virus particles were scattered in the nuclei and in the cytoplasm of secretory cells (Fig. 4E and F). Fig. 4E depicts the morphological differences of the virus in the nucleus and in the cytoplasm; in the nucleus, the virus replicated and assembled its nucleocapsid, and with the transition into the cytoplasm, the viral particles further developed. The high-magnification micrograph in Fig. 4G shows longitudinal and cross sections of the SGH virus particles in the cytoplasm of the secretory cells. In the normal salivary glands, with the transition from the secretory to the reabsorptive and then to the proximal region, the diameter of the gland decreased (Fig. 2A). The reabsorptive region was localized in the thorax of the insect, and at the electron microscope level this region was characterized by an external basal lamina with a smooth outside surface and underlying epithelial
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Fig. 1. Light and scanning electron microscopy (SEM) of the general organization of healthy salivary glands in G. pallidipes. (A) The salivary glands are characterized by three distinct regions: secretory, reabsorptive, and proximal. Bar = 100 lm. (B nad C) Light microscopy magnifications of the proximal and reabsorptive regions. Bars = 20 lm. (D and E) SEM of the secretory region of the salivary glands. Micrographs show the longitudinal muscle fibers surrounding this region of the salivary glands. Bars = 50 lm and 10 lm, respectively.
cells showing an infolded basal membrane (Fig. 5A and B). This region was very rich in microvilli that extended from the epithelium into the lumen, and the cells were involved in the reabsorption of the lumen secretion (Fig. 5A and B). Furthermore, the secretion in the lumen of this gland region contained fewer electron-dense filaments than that of the secretory region (Fig. 5A). Finally, the morphology of the proximal region was characterized by a thin basement membrane that surrounded an epithelial layer. The epithelial cells were separated from the lumen by a cuticle consisting of a light layer (endocuticle) and a dense, dark, thin layer (epicuticle) (Fig. 5C). Infection with SGH virus also affected the reabsorptive region, where the ultrastructural analysis showed a profound change of the internal organization of tissues. The epithelium appeared stratified and vacuolated, and it was difficult to distinguish the bound-
ary between epithelium and lumen (Fig. 6A). The reabsorptive cells had enormous cytoplasmic vacuoles and the microvilli appeared destroyed or damaged (Fig. 6A). In the reduced glandular lumen, it was possible to identify viral particles (Fig. 6B). The basement membrane was thicker and less compact than that of the same region of healthy salivary glands (Fig. 6C). Moreover, in the reabsorptive cells, free ribosomes, scattered strands of rough endoplasmic reticulum, and numerous degenerated mitochondria were present (Fig. 6C and D). The viral infection also affected the proximal region (Fig. 2A), but duct epithelial cells of the salivary glands underwent minor changes compared with the secretory and reabsorptive cells (Fig. 6E). No viral particles were observed in this gland region. In addition, TEM observations revealed the absence of secretion in the lumen (Fig. 6E).
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Fig. 2. Light and scanning electron microscopy (SEM) of the hypertrophied salivary glands, infected by SGH virus, in G. pallidipes. (A) The hypertrophy involves the entire gland, especially the secretory region, which is entangled with tracheae in the abdomen of the fly. Bar = 200 lm. (B and C) Light microscopy magnifications of the junction between the two glands and the transition between the proximal and reabsorptive regions. Bars = 20 lm. (D and E) SEM of the secretory region of the salivary glands. The muscle tissue undergoes a profound alteration (i.e., muscle fibers lose their organization and often are interrupted or destroyed producing holes in the sheath). Bars = 100 lm and 25 lm, respectively.
4. Discussion Based on their anatomy, insect salivary glands can be divided into two groups (House and Ginsborg, 1985). Salivary glands of the higher Diptera consist of simple tubules joined to form a common duct, whereas in other insect orders the salivary glands are more complex, having a racemose appearance with ducts and acini. Several ultrastructural studies of dipteran salivary glands have been published (House and Ginsborg, 1985; Janzen and Wright, 1971; Martoja and Ballan-Dufrançais, 1984; Wright, 1969).
In this study, a detailed reconstruction of the general organization of the salivary glands of G. pallidipes was obtained by both SEM and TEM analyses. Along the length of this organ, we could distinguish three regions, which differed in their morphology and ultrastructure: a secretory, a reabsorptive, and a proximal region. A similar division of the salivary glands was observed in other insects, as in Calliphora, where the ultrastructural and physiological observations showed a secretory distal region and a proximal reabsorptive tract (Oschman and Berridge, 1970).
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Fig. 3. Light and transmission electron micrographs of the secretory region in the normal, uninfected salivary glands. (A and B) Light and transmission electron micrographs, respectively, showing the muscular sheath that surround the glands, an epithelium composed of a single layer of cells, and a central lumen. Bars = 10 lm and 1 lm, respectively. (C) The muscular sheath shows the muscle fibers and it is separated from the epithelium by the basement membrane. Bar = 250 nm. (D) Magnification of a secretory cell showing the rough endoplasmic reticulum. Bar = 200 nm. (E) This transmission electron micrograph of the secretory cell depicts the presence of microvilli that extend into the lumen, a septate desmosome () in the apical cell membrane, and a nucleus. The lumen contains an electron-dense matrix, where there are numerous electron-opaque filaments surrounded by clear zones. Bars = 500 nm, () 50 nm. (F and G) The secretory cell contains Golgi complexes, mitochondria, and secretory granules. Bars = 50 nm and 100 nm, respectively. (H) Magnification of microvilli involved in the luminal secretion and cross sections of the electron-opaque filaments surrounded by clear zones. Bar = 100 nm. Bm, basement membrane; Ep, epithelium; Gc, Golgi complexes; Lu, lumen; M, mitochondria; Mc, muscular coat; Mv, microvilli; N, nucleus; Rer, rough endoplasmic reticulum; Sg, secretory granules.
SEM and TEM investigation of the secretory region of the G. pallidipes salivary glands allowed an accurate description of the morphology and ultrastructure of a thick muscle tissue sheath, composed of longitudinal muscular fibers that covered this region. This muscle layer may be responsible for the contraction necessary to expel the saliva, as described in other insects (Reis et al., 2003). The presence of external muscle tissue in the salivary glands varies among insects. In fact, salivary glands of Anopheles stephensi (Wright, 1969), Aedes aegypti (Janzen and Wright, 1971), and of Calliphora (Oschman and Berridge, 1970) show no muscle covering. The same is true for the salivary glands of adult Ceratitis capitata, and it has been suggested that the actin filament network may generate peristaltic contractions that move the saliva along the lumen (Riparbelli et al., 1994). Nevertheless, in the salivary glands of
other insects muscle tissue has been observed, as in Cimex hemipterus (Serrão et al., 2008) and in Triatoma infestans (Reis et al., 2003). In previous studies on tsetse flies, very little attention was paid to the involvement of the muscle sheath in the secretion process and how the secretion is discharged from the extracellular cavity. No study has focused on the normal secretory process of the salivary glands of tsetse flies even though basic information about salivary gland secretion is available from a model dipteran (Berridge and Patel, 1968; Hansen Bay, 1978; House and Ginsborg, 1985). Van Den Abbeele et al. (2010, 2007) examined the molecular aspects of the sialome of tsetse to determine the constituents of saliva, especially in relation to possible changes in the composition of the secretion caused by infection with trypanosomes, but the overall physiology appears to have been ignored.
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Fig. 4. Light and transmission electron micrographs of the secretory region in the hypertrophied salivary glands, infected by SGH virus. (A and B) Light and transmission electron micrographs, respectively, showing the alteration of the muscular sheath, the vacuolization of the epithelium, and the significantly reduced lumen. Bars = 20 lm and 2 lm, respectively. (C) In the muscle, the arrangement of muscle fibers undergoes a profound alteration and the basement membrane is degenerated. Bar = 1 lm. (D) This magnification of the epithelium shows the cell proliferation induced by the viral infection. Bar = 5 lm. (E and F) The viral particles replicate and form their nucleocapsids in the nucleus and then undergo a further development in the cytoplasm of the secretory cells. Bars = 200 nm and 100 nm, respectively. (G) High magnification of the viral particles in the cytoplasm. Bar = 100 nm. Bm, basement membrane; Ep, epithelium; Lu, lumen; Mc, muscular coat; N, nucleus; Vp, viral particles.
The secretory cells of the salivary glands of G. pallidipes contained a high number of large mitochondria that had electrondense granules, characteristic of secretory cells. These cell types possessed an extensive rough endoplasmic reticulum, Golgi complexes and numerous secretory granules, suggesting an important role of these cells in the secretion of enzymes, ions and water, as described in previous studies (House and Ginsborg, 1985; Kokwaro et al., 1991). Studies performed on Calliphora suggest that the secretory granules contain amylase, the only enzyme that has been found in the saliva of this insect. These secretion granules are probably formed in the Golgi complex, a process that has been documented clearly in other studies (Oschman and Berridge, 1970). The reabsorptive region of the G. pallidipes salivary glands was devoid of a muscular layer and the basement membrane was in direct contact with hemolymph, similar to the reabsorptive portion of the salivary glands of Calliphora (Oschman and Berridge,
1970). The basal domain of the plasma membrane of the epithelial cells was infolded, a large number of microvilli extended from the epithelium into the lumen, and the secretion in the lumen appeared more dilute than that of the secretory region, suggesting an active reabsorption in this region and a change in the composition of the saliva. Morphological and physiological studies performed on Calliphora support this hypothesis; epithelial cells of this region are involved in the reabsorption of ions, transforming the luminal fluid into a hypo-osmotic final saliva (Oschman and Berridge, 1970; Rotte et al., 2008). The proximal duct of the salivary glands was poorly investigated in tsetse flies (Kokwaro et al., 1991) and in other insects. Our results demonstrated that epithelial cells produced a cuticle whose morphology resembled that described in the crop of Phormia regina, an important organ of the alimentary tract of insects (Stoffolano et al., 2010) and indicative of foregut organization.
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Fig. 5. Transmission electron micrographs of the reabsorptive and proximal regions of the healthy salivary glands. (A and B) The reabsorptive region is bordered by a basement membrane, which is in direct contact with the hemolymph. This region shows a large number of microvilli that extend from the epithelium into the lumen and are involved in the reabsorption of the luminal secretion. Bars = 1 lm and 500 nm, respectively. (C) The proximal region is characterized by a thin basement membrane and an epithelial layer. The epithelial cells are separated from the lumen by a cuticle that consists of an internal endocuticle and an external epicuticle. Bar = 1 lm. Bm, basement membrane; Enc, endocuticle; Ep, epithelium; Epc, epicuticle; Lu, lumen; Mv, microvilli.
Important results of this study were obtained from the analysis of the profound morphological and ultrastructural changes of the G. pallidipes salivary glands, caused by the SGH virus. The viral infection produced a significant hypertrophy throughout the entire gland, but the highest degree of this hypertrophy affected the secretory region. Our SEM and TEM observations of this region showed severe alterations of the muscular coat and muscle cells, although viral particles were not detected in these cells. The
Fig. 6. Transmission electron micrographs of the reabsorptive and proximal regions in hypertrophied salivary glands. (A) The reabsorptive region shows a degenerated ultrastructure; the epithelium is vacuolated and the microvilli appear destroyed. Bar = 2 lm. (B) In the reduced gland lumen few viral particles are present. Bar = 250 nm. (C) The basement membrane is thicker and more stratified than the same region of the healthy salivary glands. Bar = 200 nm. (D) The reabsorptive cells are altered and show degenerated mitochondria. The arrow indicates scattered strands of rough endoplasmic reticulum. Bar = 100 nm. (E) A lesser degree of ultrastructural alteration seems to affect the proximal region of the salivary glands, where no viral particles were observed. Bar = 500 nm. Bm, basement membrane; Enc, endocuticle; Ep, epithelium; Epc, epicuticle; Lu, lumen; M, mitochondria; Vp, viral particles.
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longitudinal muscle fibers lost their organization, suggesting a degeneration of the myofibrils, which in normal muscle structure remain intact for proper function. In addition, the underlying basement membrane was irregular, appeared striated, and the changes of its relationships with muscle cells and proliferating epithelial cells suggest a degeneration of cytoskeletal elements that affect communication of the basement membrane with the surrounding cells. Based on these results, we can hypothesize that the contractile capacity of the muscle sheath that covers the gland is greatly compromised, which should lead to a decreased ability to expel saliva. This hypothesis is supported further by the reduction of the volume of the glandular secretion in the infected salivary glands. The disease pathology also affected the reabsorptive region of the salivary glands, where the epithelial cells were rich in degenerated mitochondria and free ribosomes, and several strands showed interruption of the rough endoplasmic reticulum, denoting a possible altered cellular activity. Moreover, our ultrastructural observations showed the lack of microvilli in this region, suggesting a compromised reabsorptive function of the infected salivary glands. Thus, although it is not known whether the enzyme composition and/or functions of hypertrophied salivary glands are affected negatively by the disease pathology, the results obtained in this study highlight the morphological and ultrastructural changes of the whole salivary gland, which are viral-induced and could be related to an overall alteration of gland function. These comments are essential to correlate the action of the SGH virus to fly infection with Trypanosoma spp. In the normal parasite infection, trypanosome development takes place in the salivary glands of tsetse flies. In particular, four sequential stages of development can be recognized in the gland: (1) the uncoated epimastigote trypanosomes (with a prenuclear kinetoplast) attached by elaborate flagellar outgrowths that ramify between host microvilli; (2) uncoated premetacyclic trypomastigotes (with postnuclear kinetoplast) and reduced flagellar outgrowths; (3) nascent metacyclic trypomastigotes that have acquired the surface coat and lost their flagellar outgrowths but remain attached to microvilli; (4) unattached coated metacyclics lying free in the saliva ready for discharge when the fly bites (Tetley and Vickerman, 1985). Previous studies reported that the impact of SGHV infection on trypanosome transmission is unclear (Burtt, 1945; Otieno et al., 1980; Whitnall, 1934). Kokwaro et al. (1991) examined the mixed infection of tsetse salivary glands, evidencing a severe cellular disintegration and a significant degeneration of trypanosomes in these cells. Based on the results obtained in this study, we can hypothesize that the ultrastructural alterations of the glandular epithelium, caused by viral infection, could impair the integrity of the structures necessary to the development and survival of trypanosomes in the salivary glands. Therefore, this observation may in part explain the degeneration of trypanosomes, as described above, and we can assume that the SGH virus could affect the normal ability of viral infected tsetse flies to transmit trypanosomes. Nevertheless, further studies are necessary to elucidate the mode of action of this virus, especially in relation to the possible physiological changes of the salivary glands, to better understand how to best manage the SGH virus in tsetse flies, which are important vectors of trypanosomes, and to explore alternative ways to eliminate the virus from the colonies being maintained for sterile insect control using massive release programs.
Disclosures Each author of this paper have not any involvement, financial or otherwise, that might potentially bias their work.
Acknowledgments We gratefully acknowledge the financial support for this study provided by the research Contract No. 16419 with International Atomic Energy Agency (IAEA). References Abd-Alla, A., Vlak, J.M., Bergoin, M., Maruniak, J.E., Parker, A., Burand, J., Jehle, J.A., Boucias, D., 2009. Hytrosaviridae: a proposal for classification and nomenclature of a new insect virus family. Arch. Virol. 154, 909–918. Abd-Alla, A., Bossin, H., Cousserans, F., Parker, A., Bergoin, M., Robinson, A., 2007. Development of a non-destructive PCR method for detection of the salivary gland hypertrophy virus (SGHV) in tsetse flies. J. Virol. Method 139, 143–149. Berridge, M.J., Patel, N.G., 1968. Insect salivary glands: stimulation of fluid secretion by 5-hydroxytryptamine and adenosine-30 , 50 -monophosphate. Science 162, 462–463. Burtt, E., 1945. Hypertrophied salivary glands in Glossina: evidence that G. pallidipes with this abnormality is peculiarly to trypanosome infection. Ann. Trop. Med. Parasitol. 39, 11–13. Feldmann, U., 1994. Guidelines for the rearing of tsetse flies using the membrane feeding technique. in: Ochieng’-Odero, J.P.R. (Ed.), Techniques of Insect Rearing for the Development of Integrated Pest and Vector Management Strategies. ICIPE Science Press, Nairobi, Kenya, pp. 449–471. Gooding, R.H., Feldmann, U., Robinson, A.S., 1997. Care and maintenance of tsetse colonies. In: Crampton, J.M., Beard, C.B., Louis, C. (Eds.), The Molecular Biology of Insect Disease Vectors: A Methods Manual. Chapman & Hall Ltd., London, UK, pp. 41–55. Hansen Bay, C.M., 1978. Control of salivation in the blowfly Calliphora. J. Exp. Biol. 75, 189–201. House, C.R., Ginsborg, B.L., 1985. Salivary gland. in: Kerkut, G.A., Gilbert, L.I. (Eds.), Comprehensive Insect Physiology, Biochemistry and Pharmacology, vol. 11. Pergamon Press, New York, pp. 195–224. Jaenson, T.G.T., 1978. Virus-like rods associated with salivary gland hyperplasia in tsetse, Glossina pallidipes. Trans. Roy. Soc. Trop. Med. Hyg. 72, 234–238. Janzen, H.G., Wright, K.A., 1971. The salivary glands of Aedes aegypti (L.): an electron microscope study. Can. J. Zool. 49, 1343–1345. Jura, W.G.Z.O., Otieno, L.H., Chimtawi, M., 1989. Ultrastructural evidence for transovum transmission of the DNA virus of tsetse Glossina pallidipes (Diptera: Glossinidae). Curr. Microbiol. 18, 1–4. Kokwaro, E.D., Otieno, L.H., Chimtawi, M., 1991. Salivary glands of the tsetse Glossina pallidipes Austen infected with Trypanosoma brucei and virus particles: ultrastructural study. Insect Sci. Appl. 12, 661–669. Kokwaro, E.D., Nyindo, M., Chimtawi, M., 1990. Ultrastructural changes in salivary glands of tsetse, Glossina morsitans morsitans, infected with virus and rickettsialike organisms. J. Invertebr. Pathol. 56, 337–346. Lietze, V.U., Abd-Alla, A.M.M., Vreysen, M.J.B., Geden, C.J., Boucias, D.G., 2011. Salivary gland hypertrophy viruses: a novel group of insect pathogenic viruses. Ann. Rev. Entomol. 56, 63–80. Martoja, R., Ballan-Dufrançais, C., 1984. The ultrastruscture of the digestive and excretory organs. in: King, R.C., Akai, H. (Eds.), Insect ultrastructure, vol. 2. Plenum Press, New York, pp. 199–261. Oschman, J.L., Berridge, M.J., 1970. Structural and functional aspects of salivary fluid secretion in Calliphora. Tissue Cell 2 (2), 281–310. Otieno, L.H., Kokwaro, E.D., Chimtawi, M., Onyango, P., 1980. Prevalence of enlarged salivary glands in wild populations of Glossina pallidipes in Kenya, with a note on the ultrastructure of the affected organ. J. Invertebr. Pathol. 36, 113–118. Reis, M.M., Meirelles, M.S., Soares, M.J., 2003. Fine structure of the salivary glands of Triatoma infestans (Hemiptera: Reduviidae). Tissue Cell 35, 393–400. Riparbelli, M.G., Callaini, G., Dallai, R., 1994. Cytoskeleton of larval and adult salivary glands of the dipteran Ceratitis capitata. Implication of microfilaments and microtubules in saliva discharge. Boll. Zool. 61, 9–17. Rotte, C., Walz, B., Baumann, O., 2008. Morphological and functional characterization of the thoracic portion of blowfly salivary glands. Arthropod Struct. Develop. 37, 372–382. Sang, R.C., Jura, W.G., Otieno, L.H., Mwangi, R.W., Ogaja, P., 1999. The effects of a tsetse DNA virus infection on the functions of the male accessory reproductive gland in the host fly Glossina morsitans centralis (Diptera; Glossinidae). Curr. Microbiol. 38, 349–354. Sang, R.C., Jura, W.G., Otieno, L.H., Mwangi, R.W., 1998. The effects of a DNA virus infection on the reproductive potential of female tsetse flies, Glossina morsitans centralis and Glossina morsitans morsitans (Diptera: Glossinidae). Mem. Inst. Oswaldo Cruz. 93, 861–864. Serrão, J.E., Castrillon, M.I., Santos-Mallet, J.R., Zanuncio, J.C., Gonçalves, T.C., 2008. Ultrastructure of the salivary glands in Cimex hemipterus (Hemiptera: Cimicidae). J. Med. Entomol. 45 (6), 991–999. Stoffolano Jr., J.G., Guerra, L., Carcupino, M., Gambellini, G., Fausto, A.M., 2010. The diverticulated crop of adult Phormia regina. Arthropod Struct. Develop. 39 (4), 251–260. Tetley, L., Vickerman, K., 1985. Differentiation in Trypanosoma brucei: host-parasite cell junctions and their persistence during acquisition of the variable antigen coat. J. Cell Sci. 74, 1–19. Van Den Abbeele, J., Caljon, G., De Ridder, K., De Baetselier, P., Coosemans, M., 2010. Trypanosoma brucei modifies the tsetse salivary composition, altering the fly
L. Guerra et al. / Journal of Invertebrate Pathology 112 (2013) S53–S61 feeding behavior that favors parasite transmission. PLoS Pathog. 6 (6), e1000926. Van Den Abbeele, J., Caljon, G., Dierick, J.F., Moens, L., De Ridder, K., Coosemans, M., 2007. The Glossina morsitans tsetse fly saliva: general characteristics and identification of novel salivary proteins. Insect Biochem. Molec. 37, 1075–1085.
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Van Den Abbeele, J., Claes, Y., Van Bockstaele, D., Le Ray, D., Coosemans, M., 1999. Trypanosoma brucei spp. Development in the tsetse fly: characterization of the post-mesocyclic stages in the foregut and proboscis. Parasitol 118, 469–478. Whitnall, A.B.M., 1934. The trypanosome infections of Glossina pallidipes in the Umfolosi game reserve, Zuhuland. Onderstepoort J. Vet. Sci. Anim. Ind. 11, 7–21. Wright, K.A., 1969. The anatomy of salivary glands of Anopheles stephensi liston. Can. J. Zool. 47, 579–587.