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T rangeli invasion in R robustus salivary


glands
ARTICLE DECEMBER 2013

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Ingrid Gracielle Martins da Silva

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Rev. Ibero-Latinoam. Parasitol. (2013); 72 (1): 31-37

Artculo de Original

In vitro and in vivo experimental infection of


Rhodnius robustus salivary glands by
Trypanosoma rangeli: An ultrastructural
approach of the initial process of invasion
BARRETO-SANTANABD1, CAETANO J. V. O.2, MARTINS DA SILVA I. G.2,
GURGEL-GONALVES R.1, NAIR BO S.2 and CUBA CUBA C. A.1

1
2

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

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.

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 Braslia, Campus Universitario Darcy Ribeiro, Asa Norte, Braslia, Distrito Federal,
Brazil.

CEP: 70910-900. Phone number: 51 61 3107-1786.

E-mail: daniellabaag@hotmail.com.

31

BARRETO-SANTANA D. et al.

RESUMEN
Algunos aspectos del proceso de penetracin del Trypanosoma rangeli en glndulas salivales de
triatominos an no estn claros. Para promover informaciones adicionales de los eventos que transcurren
durante el pasaje del parasito a travs de estas clulas glandulares, una investigacin ultra-estructural
fue realizada luego despus de experimentos in vitro e in vivo de infeccin de las glndulas salivales de
Rhodnius robustus con la cepa SC-58 de T. rangeli. Las imgenes a microscopa electrnica de barrido
mostraron que los flagelados (tripomastigotes y/o epimastigotes) se adhieren a la membrana basal de las
glndulas por los flagelos. Fue tambin documentada a presencia de flagelados adheridos en la membrana
basal de la regin cercana al ducto glandular, y las formas mayoritarias eran epimastigotes. Con una hora
de infeccin, varias agrupaciones de flagelos fueron vistos junto a los poros hecho no observado en la
superficie de la glndula de control. En la microscopa electrnica de transmisin se observ la presencia
de tripanosomas esparcidos por el lumen de la glndula. En condiciones in vitro e in vivo los flagelados
producen lesiones en la membrana basal, cruzndola y siguiendo para las clulas del epitelio glandular, en
la mayora de los casos utilizando la punta del flagelo.
Palabras clave: Rhodnius robustus, Trypanosoma rangeli, Infeccin experimental, Glndulas salivales;
Microscopa electrnica de barrido, Microscopa electrnica de transmisin.

INTRODUCTION
Trypanosoma rangeli (Kinetoplastida, Trypanosomatidae) is a protozoan flagellate, which infects several species of blood-sucking insects (Hemiptera, 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 differentiate into epimastigotes forms, which are able to
multiply and cross the intestinal epithelium. After
reaching the hemolymph, the parasites invade hemocytes, 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,

32

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 lumen 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) revealed 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 salivary 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-

Rev. Ibero-Latinoam. Parasitol. (2013); 72 (1): 31-37

TRYPANOSOMA RANGELI INVASION IN RHODNIUS ROBUSTUS SALIVARY GLANDS

mines, an investigation was conducted by at the


level of scanning electron and transmission microscopy 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 Par. 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 l of culture of strain SC58 (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 l 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

Rev. Ibero-Latinoam. Parasitol. (2013); 72 (1): 31-37

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 4C (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 specimens were infiltrated and embedded in Spurr resin. 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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BARRETO-SANTANA D. et al.

Figure 1. Scanning Electron Microscopy (SEM) of the salivary gland uninfected Rhodnius 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 Trypanosoma 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

34

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

Rev. Ibero-Latinoam. Parasitol. (2013); 72 (1): 31-37

TRYPANOSOMA RANGELI INVASION IN RHODNIUS ROBUSTUS SALIVARY GLANDS

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 experimental infection, it was observed that on the in vitro
system the flagellates appear agglomerated, appar-

Figure 9-12. Transmission Electron Microscopy from the salivary glands of


Rhodnius robustus.
Figure 9. Uninfected salivary gland of R. robustus (X24.400). Basal membrane (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 glandular 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).

Rev. Ibero-Latinoam. Parasitol. (2013); 72 (1): 31-37

35

BARRETO-SANTANA D. et al.

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 natural 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 identification and distribution of carbohydrates sugars
on the salivary glands of R. prolixus with FITClabeled lectins, the surface of salivary gland present

36

carbohydrate residues that may serve as receptors


that bind to the flagellates before the invasion. This
could suggest that in this region, flagellates recognize and damage the basal membrane more easily,
in order to cross it and reach the glandular epithelium cells. Fonseca et al. (2006) already demonstrated that lipophorins present in the hemolymph of R.
prolixus influence on the improvement of the ectoATPase 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 parasites found within salivary glands were similar to
sphaeromastigote forms. Cuba Cuba (1975) analyzing the salivary glands of R. ecuadoriensis using
optical microscopy described the presence of parasites 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 penetration of T. rangeli in the outer layer of the basal
lamina of R. domesticus salivary glands, the parasites could be found in the space between the basal
lamina and salivary gland epithelium. Epimastigotes forms were found in the basal lamina invading

Rev. Ibero-Latinoam. Parasitol. (2013); 72 (1): 31-37

TRYPANOSOMA RANGELI INVASION IN RHODNIUS ROBUSTUS SALIVARY GLANDS

the cells of the salivary gland for an unknown mechanism. 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 flagellates 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, penetrating individually through small holes. In contrast, 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 epimastigotes and those adhered in the membrane showed
characteristics of trypomastigotes. Apparently,
as suggested by Meirelles et al, (2005), epimastigotes produce a higher amount of a lytic molecule
consisting of a pore-forming protein, the so called
rangelysin allowing the passage of parasites by epithelial 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 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 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.

37