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Parasitología al día

versão impressa ISSN 0716-0720

Parasitol. día v.22 n.3-4 Santiago jul. 1998

http://dx.doi.org/10.4067/S0716-07201998000300002 

TRABAJO DE INVESTIGACION

ULTRASTRUCTURAL OBSERVATIONS ON Trypanosoma
(Herpetosoma) rangeli IN THE SALIVARY GLANDS OF
Rhodnius ecuadoriensis (Hemiptera, Reduvidae)

ELLIOT W. KITAJIMA*, CESAR A. CUBA CUBA* and ZIGMAN BRENER**

* University of Brasilia, Instituto de Ciências Biológicas; Departamento de Patologia, Unidade de Parasitologia Médica Faculdade de Ciências da Saúde Brasília D.F. 70-7910-900, Brazil; ** Instituto Renê Rachou, FIOCRUZ, Belo Horizonte, MG. Brazil.

ABSTRACT

An electron microscopy study was carried out on the salivary glands of the triatomine bug Rhodnius ecuadoriensis experimentally infected with a Peruvian strain of Trypanosoma (Herpetosoma) rangeli. Epimastigotes forms of T. rangeli were found in large numbers in the haemolymph surrounding the salivary gland. Some of them were closely associated with the basal lamina of the gland epithelium, apparently in a process of cell invasion, in which the flagellum breaking through the basal lamina. Within the cytoplasm of the gland epithelial cells, there were rounded forms of T. rangeli, consisting of tightly coiled sphaeromastigotes. The gland lumen was filled with large numbers of mostly epimastigotes forms of the flagellate; only few paramastigotes and trypomastigotes forms could be unequivocally identified. The presence of paramastigotes in the life cycle of T. rangeli has yet to be fully assessed.

Key words: Trypanosoma (Herpetosoma) rangeli, ultrastructura, salivary glands Rhodnius ecuadoriensis.

 

INTRODUCTION

Trypanosoma (Herpetosoma) rangeli, Tejera 1920 is widely distributed in Latin-America, naturally infecting many vertebrates, including man.1-3 Its pathogenic role in human beings, however, is still unclear but T. cruzi and T. rangeli sharecommon triatomine vectors and reservoirs mammals in most endemic areas making complex the epidemiology of Chagas' Disease.4

Triatominae bugs, mainly of the Genus Rhodnius, not only serve as vectors for T. rangeli, but are infected and suffer pathogenic effects by this flagellate.5

Several workers have investigated the life cycle of T. rangeli in its vector naturally and experimentally infected. From these studies it is known that the evolutionary cycle of the flagellate involves an intestinal phase, the passage to the haemolymph followed by its multiplication in the hemocytes, and culminates with the infection of the salivary glands, where the infective metacyclic trypomastigotas are formed and eventually inoculated into the vertebrate hosts during blood feeding.6,7

The invasion of the salivary glands of triatomine bugs by the flagellate was first studied, at light microscopy, by Groot,8 and it was later followed by several authors.9-12 In Rhodnius prolixus infected with T. rangeli Ellis et al,13 and Hecker et al,14 published, in our knowledge, the only available studies on salivary gland infection at electron microscopy level.

However, many aspects of the salivary gland infection process are still obscure, such as the morphology of both the intracellular forms, at cell glands and those of the salivary gland lumen contents and the process of metacyclogenesis. In order to provide further additional information regarding these points, an electron microscopic investigation was made, using R. ecuadoriensis, experimentally infected with a Peruvian strain of T. rangeli.

MATERIAL AND METHODS

Specimens of nymphs IV and V instar of R. ecuadoriensis from our laboratory colony were infected experimentally with a well characterised strain of T. rangeli through feeding on infected guinea-pigs (Cavia porcellus). Examination of faecal and haemolymph samples as described elsewhere3 showed abundant flagellates between 27-31 days after infective blood meal. Presence of the parasite at the salivary glands was proved by experiments of bite transmission of T. rangeli on outbred white mice between 8-18 days after positive haemolymph infection.

Salivary glands from these bugs were removed by dissection, approximately 45 days after the infection, and immediately fixed in 1% osmium tetroxide in 0.1M phosphate buffer, pH 7.2. After dehydration, the glands were embedded in Epon 812, and the blocks sectioned with a Du Pont diamond knife, mounted on a LKB Ultratome III ultramicrotome. Sections were stained with uranyl acetate and Reynold's lead citrate, then examined in a JEOL JEM-100C electron microscope.

RESULTS

Despite some technical limitations related to prefixation and preparation of blocks, the observations of the sections provided some valuable information, which are described below:

Accumulation of T. rangeli epimastigotes outside the salivary gland:

A large number of T. rangeli cells accumulated outside the salivary gland wall were observed reminding its description elsewhere on light microscopy. As judge by longitudinal sections, most if not all of them were epimasti-gotes, and they appeared clustered at some distance from the basal lamina of the salivary gland and associated muscle cells (Figures 1 and 2). Some of the flagellum zone of the T. rangeli cells, however, appeared to be in contact with the basal lamina but without any attachment structure (Figure 1). The salivary gland had in some places a thin muscular layer, composed of both gland and muscle cells covered by the basal lamina. In a few instances, a possible initiation of the T. rangeli penetration into the salivary gland or the adjacent muscle layer could be observed. The flagellum appeared to be penetrating into the cytoplasm through ruptured basal lamina (Figure 1, inset); however, T. rangeli cell bodies half-buried in the cytoplasm could not be seen. Penetration occurs in muscle and gland cells, since T. rangeli was observed in both cell types (Figures 3 and 4).

T. rangeli within the salivary gland epithelial cells:

Epithelial cells of the salivary gland of R. ecuadoriensis presented no remarkable features (Figures 1, 4 and 6). The gland is formed by a single layer of secretory cells, with a prominent nucleus. In the basal region these cells are covered by a thick basal lamina; sometimes the plasma membrane was invaginated. At the apical region microvilli or cilia were absent. The cytoplasm was rich in ribosomes and small mitochondria; there were few Golgi bodies, clusters of lysosome-like vesicles and poorly developed endoplasmic reticulum. Septated desmosomes appeared between adjacent cells. In salivary glands from T. rangeli- infected R. ecuadoriensis, the flagellates could be easily detected in the cytoplasm, randomly distributed (Figure 4). They usually had eliptical or rounded profiles as a result of tight coiling with their flagella that were outermost. T. rangeli cells were seen in cytoplas-mic pockets, apparently limited by a membrane which was not always clearly discernible (Figure 5). In some cases, T. rangeli cells had more than one flagellum, nucleus and/or kinetoplast. Because of the curled position, it was difficult to establish the developmental stage of T. rangeli cells within the salivary gland cells, but probably they were closer to sphaeromastigotes, and since a relatively free and long flagellum was clearly seen around the cell body (Figures 4 and 5) they certainly were not amastigotes. No special association of cell structures around T. rangeli cells, was observed in the cytoplasm of salivary gland cells.

Luminal content of the R. ecuadoriensis salivary gland infected with T. rangeli:

The lumen of the salivary gland was filled by a dense, amorphous material (Figure 6); there was also a large number of T. rangeli cells. Most of them appeared associated perpendicularly to the apical region of the salivary gland, with their flagellum usually in contact with the gland cell surface (Figures 6 and 7). No desmosome- like structure could be observed where the flagellum touched the apical cell surface. In the central region of the lumen, T. rangeli cells did not exhibit special arrangement, being scattered randomly. Analyses of the longitudinal sections of T. rangeli cells in the lumen indicated that most of them were epimastigotes. Some of them contained two nuclei, kinetoplasts and flagella, suggesting that they were in a division process. Paramastigo-te and trypomastigote forms were found only rarely (Figure 8). T. rangeli cells found in the gland lumen usually contained vesicles of varied size and number, with a dense content (Figures 7 and 8).

 

Figure 1. Periphery of the salivary gland, showing a large number of epimastigotes (EP) of T. rangeli in the haemolymph, some of them in contact with the gland basal lamina (b) through the flagellum. E: salivary gland epithelium. (10,000 X) (f) Inset shows a flagellum penetrating through ruptured basal lamina. (20,000 X).

Figure 2. Detail of an epimastigote of T. rangeli in the vicinity of the salivary gland. (c): centriole; (k): kinetoplast; (n): nucleus. (12,000X).

Figure 3. Two T. rangeli cells within the thin muscle layer (M) surrounding the salivary gland. (15,000 X).

Figure 4. A general view of part of the salivary gland epithelium (E) containing some roughly rounded forms of T. rangeli (arrows) (6,600 X).

Figure 5. Detail of two flagellates in the salivary gland cytoplasm. They appear coiled, with the flagellum (f) at the periphery, and are contained in membrane bounded cavities. (k): kinetoplast; (m): mitochondrion. (20,000 X).

Figure 6. Low magnification image of the salivary gland epithelium (E). A thin muscle layer (M) appears at the basal portion. The lumen (L) is filled with a dense amorphous secretion product and contain a large number of T. rangeli cells. (b): basal lamina; (n): nucleus. (8,000 X).

Figure 7. Flagellates, mostly epimastigotes, appear arranged perpendicular onto the apical portion of the salivary gland epithelium, flagellum foremost. (6,600 X).

Figure 8. Detail of two T. rangeli cells, one of them (right) obviously a paramastigote, and the other (left) possibly a trypomastigote form. (f): flagellum; (k): kinetoplast; (n): nucleus. (12,000X).

 

DISCUSSION

The present observations confirm most the previous findings regarding T. rangeli behaviour in the salivary gland of naturally and experimentally infected triatominae bugs.12, 13 On the other hand, because of the better resolution of the electron microscope, they added further details in under-standing the relationship between the flagellate and the salivary gland compartment.

There are indeed accumu-lation of epimastigotes in the haemolymph around the saliva-ry gland, but most of the cells were at some distance from the gland. This seemed to result from the fact that T. rangeli cells aproached the gland, flagellum foremost, thus the main cell body of most cells were kept away from the gland basal region. Differently to described by Ellis et al,13 the "giant forms" were never seen on the outer haemocoel glandular membranes.

Some evidence of the gland invasion by T. rangeli, by active disruption of the basal lamina by the flagellum was observed. But the passage of the entire flagellate cell body from the haemolymph into the gland epithelium cytoplasm could not be observed. Perhaps this process is extremely rapid and difficult to detect. Both gland and adjacent thin muscle layer are invaded by T. rangeli; the fate of the flagellates which penetrate into the muscle cells was indefinable. Their either could multiply and return to the haemolymph or further penetrate into the salivary gland. Within the cytoplasm of the salivary gland epithelial cells T. rangeli were always found in a coiled form, with the flagellum curled helically around the cell body resulting in the round profile. CUBA-CUBA12 in his studies on light microscopy suggested to be sphaeromasti-gotes as defined by Brack,15 and that seems to be most appropriate. Some of the these coiled forms had more than one nuclei, kinetoplast or flagellum suggesting that they were multiplying. Apparently the coiled forms move from the basal to apical region of the cell gland passively. It was unclear whether or not the T. rangeli cells are within a membrane-bounded cavity in the cytoplasm. In some sections, a limiting membrane were obser-ved, but in other the membrane is absent. Perhaps both situations might occur.

The association of phagocytic cells with other flagellates and protozoa involves an endocytic process and therefore, a membrane limited cavity, where the ingested cells are found. For Ellis et al,13 the trypanosomes are able to penetrate directly but have to form vacuoles which contains the flagellates and from which they escape into the celI gland. In the present case in our model, T. rangeli penetration seems to be active, involving the rupture of the basal lamina and the subjacent cell membrane, thus exposing the cytoplasm, into which the flagellates penetrate. It is likely that in some instances an endocytic process might occur in exposed but intact cell membrane, while in other, the flagellate enters the cytoplasm, being later surrounded by a membrane formed "de novo". The process through which T. rangeli are expelled from the cytoplasm into the lumen could not be observed. It might occur as an exocytotic process but an active role of the parasite at a final stages cannot be ruled out.

No elongated forms of the flagellate were present within the gland cells. The finding of such forms in light microscope studies might be an artifact derived from the use of whole mounts of the gland; some extracellular elongated forms could have been trapped in the preparation and mistakenly considered intracellular. Another possibility is that preparative process could "uncurl" some curled forms. In the lumen, T. rangeli accumulates in large numbers. Contrary to the light microscopy observations, that metacyclic trypomastigotes are the prevalent forms in the salivary gland lumen, present studies showed that epimastigotes were the dominant form, at least as evaluated in favourable sections. One possible explanation for this diverging data is that the metacyclics might become dominant in later phases of the infection, not covered by the present studies. Only few possible paramastigo-tes and trypomastigotes forms were identified. The observation of paramastigotes causes specula-tion as to whether this form is part of the metacylcogenesis process. Besides, if this form represent a stage in the life cycle of T. rangeli has yet to be fully demonstrated.

The tendency of T. rangeli cells to arrange perpendicularly onto the apical part of the gland cells possibly results from the movement of the flagellate within the lumen; T. rangeli cells moving with the flagellum ahead hit the plasma membrane of the apical part of the gland cells and are held in place. As observed by CUBA CUBA12 at light microscopy, apparently some parasites still multiply in the lumen, since cells with two nuclei, kinetoplast or flagella are occasionally found.

RESUMEN

Fué realizado un estudio ultraestructural de las glándulas salivares del triatomino Rhodnius ecuadoriensis experimentalmente infectado por el tripanosomatídeo, Trypanosoma (Herpetosoma) rangeli, con la finalidad de documentar los aspectos básicos de la infección glandular por este flagelado.

Gran número de formas epimastigotes fueron encontradas en la hemolinfa que baña la parte externa de las glándulas en el hemoceloma. Algunos flagelados parecían íntimamente asociados a la membrana basal del epitelio glandular y varios de ellos parecían estar iniciando el proceso de su invasión, rompiendo la lámina basal con el flagelo. En el interior de las células glandulares observamos que, T. rangeli se presentaba en la forma de esferomastigotes, células redondeadas rodeadas externamente por un flagelo, que envolvía apretadamente el parásito.

En la luz glandular también observamos gran número de epimastigotes mezclados con los productos de la secreción glandular; el material presentó apenas pocas formas paramastigotes y trypomastigotes identificadas de manera inequívoca. El significado de los paramastigotes en el ciclo evolutivo del T. rangeli aguarda futuros estudios.

Palabras claves: Trypanosoma (Herpetosoma) rangeli, ultraestrucura, glándulas salivares Rhodnius ecuadoriensis.

REFERENCES

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