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TrangeliinvasioninRrobustussalivaryglands

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Artículo de OriginalRev. Ibero-Latinoam. Parasitol. (2013); 72 (1): 31-37

In vitro and in vivo experimental infection ofRhodnius robustus salivary glands byTrypanosoma rangeli: An ultrastructuralapproach of the initial process of invasion

BARRETO-SANTANABD1, CAETANO J. V. O.2, MARTINS DA SILVA I. G.2,GURGEL-GONÇALVES R.1, NAIR BÁO S.2 and CUBA CUBA C. A.1

1 Laboratory of Medical Parasitology and Vector Biology, Faculty of Medicine, University of Brasilia, Brazil.2 Laboratory of Electron Microscopy, Department of Cell Biology, Institute of Biological Sciences, University of

Brasilia.

Received: 18 March 2013. Accepted: 26 June 2013.Mailing address: Laboratory of Medical Parasitology and Vector Biology, Area of Pathology, Faculty of Medicine,

University of Brasília, Campus Universitario Darcy Ribeiro, Asa Norte, Brasília, Distrito Federal, Brazil.

CEP: 70910-900. Phone number: 51 61 3107-1786. E-mail: daniellabaag@hotmail.com.

ABSTRACT

The mechanism of invasion of triatomine salivary glands by Trypanosoma rangeli remains little known. To promote further information on the initial process of parasite invasion into glandular cells, an ultrastructural investigation was conducted after in vitro and in vivo infection of Rhodnius robustus salivary glands by a Brazilian strain of T. rangeli (SC-58). The Scanning Electron Microscopy images showed that the flagellates (trypomastigotes and/or epimastigotes) adhere themselves in the basal membrane of the glands mainly by the flagellum. It was also observed agglomerate of flagellates adhering in the basal membrane near the glandular duct, mostly epimastigotes. After one hour of infection, multiple clusters of flagellates spread out near to visible pores were recorded, an event not observed on the surface of the control gland. In Transmission Electron Microscopy it was observed the presence of various cross-sections and longitudinal figures of trypanosomes filling the gland lumen. The infected glands presented parasites similar to epimastigotes and trypomastigotes adhered to the microvilli and spreaded in the lumen. Under our experimental conditions flagellates (trypomastigotes and/or epimastigotes) appear to cause lesions in the basal membrane, crossing through it and invading the cells of glandular epithelium, in most cases using the tip of the flagellum. Key words: Rhodnius robustus, Trypanosoma rangeli, Experimental infection, Salivary glands, Scanning electron microscopy, Transmission electron microscopy.

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INTRODUCTION

Trypanosoma rangeli (Kinetoplastida, Try-panosomatidae) is a protozoan flagellate, which in-fects several species of blood-sucking insects (He-miptera, Reduviidae, Triatominae) and mammals, including man. T. rangeli is widely distributed in South and Central America, often overlapping its geographical distribution with that of Trypanosoma cruzi, the etiologic agent of Chagas disease (Cuba Cuba, 1998, Grisard et al, 1999). Briefly, the life cycle of T. rangeli in triatomines (mainly Rhodnius species) begins with the intake of blood trypomastigotes from infected vertebrate hosts. The parasites then colonize the digestive tract of insects, adhere in the intestinal wall and differ-entiate into epimastigotes forms, which are able to multiply and cross the intestinal epithelium. After reaching the hemolymph, the parasites invade he-mocytes, multiply and migrate infecting the bug salivary glands (Cuba Cuba, 1998). Nevertheless the developmental stages responsible for the penetration into the gland and the mechanisms involved in this process remain little known (Meirelles et al, 2005). The invasion of the salivary glands of triatomines by T. rangeli was first studied in level of optical microscopy (Groot, 1952; Herrer, 1964; Watkins, 1971; Cuba Cuba, 1975). Few studies have been pursued using electron microscopy, among them,

Ellis et al. (1980) and Hecker et al. (1990) carried on studies of the course of infection of T. rangeli in Rhodnius prolixus salivary glands and Kitajima et al. (1998) conducted further research in Rhodnius ecuadoriensis. The crossing of the parasites, from intestinal lu-men to the hemocel, and from this compartment in the lumen of the salivary gland, are critical steps in the life cycle of T. rangeli in the invertebrate host. Ultrastructural studies on penetration of T. rangeli into the cells of the middle intestine of its vectors (Hecker et al, 1990; Oliveira & Souza, 2001) re-vealed important and meaningful contributions to the biology of T. rangeli. Deserve special mention the outstanding observations by Ellis et al. (1980) on the events disclosed by the trypanosome into the sal-ivary glands of experimentally infected R. prolixus. There is still disagreement about the mechanisms used by trypanosomes to penetrate and cross the epithelium. Watkins (1971) reported the presence of damaged areas in the intestinal epithelium of R. prolixus infected with T. rangeli. Oliveira & Souza (2001) suggested that the parasite crosses the cytoplasm of intestinal cells, causing cell damage. However, it was also proposed that the T. rangeli crosses the intestinal barrier via an intracellular pathway without damaging the cells (Hecker et al, 1990). To promote additional information on invasion of T. rangeli through glandular cells of the triato-

RESUMEN

Algunos aspectos del proceso de penetración del Trypanosoma rangeli en glándulas salivales de triatominos aún no están claros. Para promover informaciones adicionales de los eventos que transcurren durante el pasaje del parasito a través de estas células glandulares, una investigación ultra-estructural fue realizada luego después de experimentos in vitro e in vivo de infección de las glándulas salivales de Rhodnius robustus con la cepa SC-58 de T. rangeli. Las imágenes a microscopía electrónica de barrido mostraron que los flagelados (tripomastigotes y/o epimastigotes) se adhieren a la membrana basal de las glándulas por los flagelos. Fue también documentada a presencia de flagelados adheridos en la membrana basal de la región cercana al ducto glandular, y las formas mayoritarias eran epimastigotes. Con una hora de infección, varias agrupaciones de flagelos fueron vistos junto a los poros hecho no observado en la superficie de la glándula de control. En la microscopía electrónica de transmisión se observó la presencia de tripanosomas esparcidos por el lumen de la glándula. En condiciones in vitro e in vivo los flagelados producen lesiones en la membrana basal, cruzándola y siguiendo para las células del epitelio glandular, en la mayoría de los casos utilizando la punta del flagelo. Palabras clave: Rhodnius robustus, Trypanosoma rangeli, Infección experimental, Glándulas salivales; Microscopía electrónica de barrido, Microscopía electrónica de transmisión.

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mines, an investigation was conducted by at the level of scanning electron and transmission mi-croscopy after transmission experiments using in vitro and in vivo infection of the salivary glands of Rhodnius robustus, recording their interaction with a Brazilian strain of T. rangeli.

MATERIAL AND METHODS

Biological Material. Specimens of R. robustus were obtained from colonies maintained at the Laboratory of Medical Parasitology and Vector Biology (LPMBV), Faculty of Medicine, University of Brasilia (UnB), originally collected in the city of Maraba, State of Pará. Details of the methods used for the maintenance of insect colonies were described elsewhere (Barreto-Santana et al, 2011). The parasites used for experimental infections were from a Brazilian strain of Trypanosoma rangeli (SC-58) which was isolated by Steindel et al. (1991), from the wild rodent, Echimys dasythrix (Grisard et al., 1999) and kept cryopreserved in the Dermatology Laboratory of UnB.

Salivary glands in vitro infection. Twenty adult male R. robustus were unfed for a period of one month before their salivary glands removed. For control, a salivary gland was extracted and placed directly in fixative for processing. For experimental salivary gland infection, firstly 500 µl of culture of strain SC-58 (2.8 x 107) was washed twice in PBS, pH 7.2 by centrifugation. Then, the supernatant was discharged and on it was added 500 µl of buffer (200 mM NaCl, 5.4 mM KCl, 1 mM CaCl2, 2 mM NaHCO3, pH 6.8), allowing the incubation for gland infection in four established times: 30 minutes, 1 hour, 3 hours and 24 hours (Oliveira & Souza, 2001).

Salivary glands in vivo infection. Artificial repasts were performed with 25 R. robustus nymphs of 4th and 5th stages. The feeding meal consisted of 3 ml of a culture of strain SC-58 (2.8 x 107) mixed with 5 ml of human blood in tubes with heparin. The meal was heated and the container adapted to a device designed for artificial xenodiagnoses purpose. Then, the nymphs were allowed to perform the infectious blood meal. Fourteen days later, intestinal contents and haemolymphatic samples were collected to confirm the infection. Since there

was no success in transmission by bites, glands were extracted about 45 days after the infective meal, cutting the dorsal thoracic muscles and fat bodies of the insect, and carefully pulling the head along with the glands attached (Cuba Cuba, 1975). After extraction, it was confirmed the presence or not of living forms of T. rangeli in the glands by optical microscopy.

Scanning Electronic Microscopy (SEM). At the end of the infection time, and confirmation of in vivo infection, the glands were extracted, fixed overnight at 4°C (2% glutaraldehyde, 2% paraformaldehyde and 3% sucrose), washed in sodium cacodylate buffer 0.1 M, pH 7.2 and post-fixed for 1 hour in 0.8% potassium ferricyanide and 1% osmium tetroxide in the same buffer. After the samples were dehydrated in ascending series of acetones, brought to the drying apparatus to the critical point of Balzers CPD 30, mounted on stubs and metalized on the device Sputter Coater, Balzers SCD 050. The samples were observed with scanning electron microscopes JEOL JEM 840A and JEOL JSM 7001F.

Electronic Transmission Microscopy (TEM). The glands were fixed, post fixed and dehydrated in acetone, as previously described for scanning electron microscopy. After dehydration the speci-mens were infiltrated and embedded in Spurr res-in. Ultrathin sections were contrasted with uranyl acetate and lead citrate, and examined under a transmission electron microscope JEOL 1011.

RESULTS

Invasion of the salivary glands of Rhodnius robustus by T. rangeli in Scanning Electron Microscopy observations. The salivary gland used as a control presented a smoothly surface of basal lamina, but with small visible wrinkles and small cracks (Figure 1). With 30 minutes of infection, the gland showed in the area near the duct, an accumulation of flagellates attached by the flagellum. There was also the presence of a group of flagellates near a narrow aperture slot in the basal lamina (Figure 2). After one hour post-infection larger number of flagellates covered the membrane apparently attached to its surface. After three hours, the

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Figure 1. Scanning Electron Microscopy (SEM) of the salivary gland uninfected Rhod-nius robustus (X140). Arrows indicate small fractures in the basal lamina (X1.600).Figure 2. SEM of R. robustus salivary gland after 30 minutes of infection by Trypano-soma rangeli (X110). In A, flagellates in the basal lamina near the duct (X3.000). In B, flagellates present in the basal lamina near a slit (arrow) (X1.400).Figure 3. T. rangeli covering the basal membrane of the salivary gland of R. robustus after 24 hours pos-infection (X80). Indent highlighting the gland surface entirely covered by flagellates (X190).

Figure 4-8. Scanning electron microscopy from the salivary glands of R. robustus infected by T. rangeli (SC-58), in vivo infection.Figure 4. Epimastigotes of T. rangeli adhered by flagellum (X3.300).Figure 5. Trypomastigote adhered to the basal membrane. Flagellar pocket (Fp) undulating membrane (Um) (X8.500).Figure 6. Salivary gland of R. robustus (X60). Highlighted the accumulation of flagellates (arrows) near the salivary duct (X2.200).

Figure 7. Flagellates adhered to the basal membrane by the middle part (arrow) of the flagellum (X2.700).Figure 8. Flagellates adhered to basal membrane by the posterior part (arrow) (X6.000). Flagellar pocket (Fp).

infected gland presented clusters of flagellates spreaded by the membrane near large number of slits and others attached near the glandular duct, as observed previously in the gland after 30 minutes of infection. There was also the presence of flagellates free and loosed from attachment of the basal membrane with morphological characteristics

of long trypomastigotes holding an undulating membrane starting from the posterior end. At twenty four hours post-infection, the salivary gland was morphological altered, but in spite of that was fully covered by flagellates, eluding the identification of the basal lamina (Figure 3). Comparing with the observations results by in

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vitro infections, the presence of trypomastigotes and epimastigotes were observed adhering the gland basal membrane (Figures 4 and 5). It was registered the presence of clusters of flagellates near of the glandular duct (Figure 6). The images show the apparent penetration through the glandular basal membrane, by the parasite middle region of the body (Figure 7) or by the tip of the flagellum (Figure 8). The latter event was not observed during the in vitro experiments.

Invasion of the salivary glands of Rhodnius robustus by T. rangeli in Transmission Electron Microscopy. The control gland presented sections of the basal membrane, secretory cells with prominent nucleus, cytoplasm rich in ribosomes, small mitochondrion and filled material into the gland lumen (Figure 9). Following the 30 minutes of infection, it was observed the presence of various transverse and longitudinal sections of trypanosomes spread in the gland lumen (Figure 10). It was possible to identify some forms and all presented characteristics of

epimastigotes (Figure 11). After one and three hours of infection, there were no spread trypanosomes in the glands. The images showed only glandular cellular structures in spite of attempts using several semi-fine and ultra-thin sections searching for the presence of parasites. The gland with 24 hours of infection could not be processed because its original form was compromised by remaining in a very long period of incubation. The glands that went through the in vivo process of infection presented forms with features of epimastigotes and trypomastigotes. In some regions it was observed the presence of many parasites, some of them possible adhered to microvilli and others free dispersed in the lumen (Figure 12).

DISCUSSION

Comparing the two different methods of experi-mental infection, it was observed that on the in vitro system the flagellates appear agglomerated, appar-

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Figure 9-12. Transmission Electron Microscopy from the salivary glands of Rhodnius robustus.Figure 9. Uninfected salivary gland of R. robustus (X24.400). Basal mem-brane (Bm), mitochondrion (Mt) and lumen (L).Figure 10. Salivary gland of R. robustus infected with T. rangeli (SC-58) after 30 minutes (X72.900). It is observed in a cross section the nucleus (N) of the parasite, and in longitudinal cross, the region with the kinetoplast (K) and flagellum (F).Figure 11. Epimastigote found after 30 minutes of infection, adhered to glan-dular cell by flagellum (X24.400). Nucleus (N), kinetoplast (K), flagellum (F), basal membrane (Bm).Figure 12. Salivary gland of R. robustus infected by T. rangeli (SC-58) via artificial repast (X10.100). There are several parasites dispersed and adhered to the membrane, among them, an epimastigote (Ep) and trypomastigote (Tr). Nucleus (N), kinetoplast (K), flagellum (F).

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ently trying to cross the basal membrane by a single hole. On the other hand, on the in vivo system they appear isolated, suggesting that on an individual basis, they penetrate by small orifices present in the basal membrane, as described by Meirelles et al. (2005). We do not have a good explanation for the above events, but they presumably reflect two natu-ral behaviors of T. rangeli in its vectors. Active disruption of the salivary gland basal lamina by the T. rangeli flagellum was previously observed (Kitajima et al, 1998), but the entire passage of the body of the flagellate were never observed, probably because it is an extremely fast and difficult process to detect. They also observed that the penetration of T. rangeli occurred in an active form involving disruption of the basal lamina, exposing the cytoplasm and facilitating the penetration of the flagellum. During our observations it was verified some disruption of the basal membrane, which in most cases were caused by individual flagellates. Oliveira & Souza (2001) showed a little quantity of T. rangeli flagellates adhered on epithelial cells of R. prolixus. But they also observed aggregations of flagellates in the same cell, usually resulting in damage to those cells. This study showed that flagellate attacked only a few epithelial cells and they suggest that this probably occurred because certain cells are recognized by flagellates to further attack and invasion. In both type of infections analyzed in SEM it was found an agglomerate of flagellates present in the basal membrane region near the glandular duct, and in the majority of instances with characteristics of epimastigotes. The flagellates (trypomastigote and/or epimastigote) are directed and recognize the tissue of basal membrane glands, and epimastigote and/or trypomastigote are adhered by flagellum, which was verified in the first 30 minutes of exposure in the in vitro experimental design. After one hour of infection, several groups of flagellate are seen near the pores. Those pores are not seen on the surface of the control gland. This suggest that the flagellum damages on the basal membrane, and forcing damages on the entrance of the glandular epithelium cells, probably caused by the tip of the flagellum.According Basseri et al. (2002), in a study of iden-tification and distribution of carbohydrates sugars on the salivary glands of R. prolixus with FITC-labeled lectins, the surface of salivary gland present

carbohydrate residues that may serve as receptors that bind to the flagellates before the invasion. This could suggest that in this region, flagellates recog-nize and damage the basal membrane more easily, in order to cross it and reach the glandular epitheli-um cells. Fonseca et al. (2006) already demonstrat-ed that lipophorins present in the hemolymph of R. prolixus influence on the improvement of the ecto-ATPase activity probably acting on the mechanism of tissues invasion of the parasites. Dos-Santos et al. (2012) revealed the presence of phosphotyrosin protein in salivary gland extract of R. prolixus. They cited that the dephosphorylation of phosphotyrosine residues of this protein associated with the membrane have an important function on the binding of T. rangeli to salivary gland cells, modifying the physiology and cell interactions due to an alteration of the cytoskeleton. These facts facilitate the setting of long epimastigotes after rupture of the cell surface. Our results also showed groups of epimastigotes attaching themselves to the same region of the gland, indicating that some parasites take advantage probably by the action of dephosphorylation of neighbors specimens within the clusters acting in a cooperative manner of attack before the invasion. Ellis et al. (1980) studying the invasion of the salivary glands of R. prolixus by T. rangeli suggested that flagellates penetrate the salivary gland cells by a process of endocytosis, and are enclosed within vacuoles. Then the parasite penetrates on the lumen of gland, where most parasites appear to lose their vacuoles before leaving the cells. Indeed, our study showed parasites in the lumen of the host gland without the vacuoles. Kitajima et al. (1998) showed that the para-sites found within salivary glands were similar to sphaeromastigote forms. Cuba Cuba (1975) ana-lyzing the salivary glands of R. ecuadoriensis using optical microscopy described the presence of para-sites which were identified as sphaeromastigotes within the cytoplasm of the glandular cells. In the present paper it was not possible to identify these sphaeromastigote forms. According to Meirelles et al. (2005), after pe-netration of T. rangeli in the outer layer of the basal lamina of R. domesticus salivary glands, the para-sites could be found in the space between the basal lamina and salivary gland epithelium. Epimasti-gotes forms were found in the basal lamina invading

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the cells of the salivary gland for an unknown mech-anism. After reaching the gland lumen, the parasites appear as epimastigotes and continue to be attached by the flagella to the microvilli of the salivary gland cells. Similarly, in our study, it was observed flagel-lates with epimastigote characteristics, both in the space between the basal lamina and the epithelium of the salivary gland, and in the lumen of the gland, the cells were adhered to the microvilli. In TEM, Meirelles et al. (2005) demonstrated that the flagellum of epimastigotes always appear arranged in the basal lamina of salivary gland, pe-netrating individually through small holes. In con-trast, in the in vitro infection, there was penetration of various aggregates of flagellates by larger and visible holes. Flagellates found penetrating in the basal membrane exhibit characteristics of epimas-tigotes and those adhered in the membrane showed characteristics of trypomastigotes. Apparently, as suggested by Meirelles et al, (2005), epimasti-gotes produce a higher amount of a lytic molecule consisting of a pore-forming protein, the so called rangelysin allowing the passage of parasites by epi-thelial barriers. These interpretations cited by the authors deserve further in-depth studies to define more clearly the molecular or biochemical basis of this important mechanism of glandular invasion. In our evaluation under in vitro and in vivo experi-mental conditions flagellates (trypomastigotes and/or epimastigotes) appear to cause lesions in the bas-al membrane, crossing through it and invading the cells of glandular epithelium, in most cases using the tip of the flagellum as an element of invasion.

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Acknowledgements: To the CNPq, CAPES and FINEP for financial support. To the Students of the Electron Microscopy Laboratory of Brasilia University for their collaboration in the preparation and analysis of samples from Scanning and Transmission Electron Microscopy and to Shigueru Ofugi and Walcymar P. Santiago, from the Laboratory of Chagas Disease (NMT-UNB) for their support in using the apparatus of Artificial Feeding of triatomines; to Maria Irani N. Barbosa and Yolanda Nascimento for their support in translation; and the anonymous reviewers for their criticisms and suggestions.