Experimental Parasitology 103 (2003) 102–111

www.elsevier.com/locate/yexpr

In vivo and in vitro metacyclogenesis tests of two strains

of Trypanosoma cruzi in the triatomine vectors
Triatoma pseudomaculata and Rhodnius neglectus:
short/long-term and comparative studyq
C.J. Carvalho-Moreira,a M.C.D. Spata,a J.R. Coura,a E.S. Garcia,b P. Azambuja,b
M.S. Gonzalez,c and C.B. Melloc,*
a
Department of Tropical Medicine, Oswaldo Cruz Institute, FIOCRUZ, Av. Brasil 4365, Rio de Janeiro, CEP 21045, RJ, Brazil
b
Department of Biochemistry and Molecular Biology, Oswaldo Cruz Institute, FIOCRUZ, Av. Brasil 4365, Rio de Janeiro, CEP 21045, RJ, Brazil
c
Department of General Biology, Universidade Federal Fluminense, Morro do Valonguinho s/n, Cx. Postal 100436, CEP 24.001-970, Niter

oi, RJ, Brazil
Received 12 April 2002; received in revised form 11 April 2003; accepted 14 April 2003

Abstract

Metacyclogenesis of Trypanosoma cruzi of the Y and Berenice strains was studied in Triatoma pseudomaculata and Rhodnius
neglectus. Results in vivo showed a higher production of metacyclic trypomastigotes in R. neglectusÕ digestive tube than in T.
pseudomaculata. In vitro experiments were also carried out in order to compare the behavior of culture forms of T. cruzi incubated in
extracts of different compartments (stomach, intestine, and rectum) of the digestive tract of both species of triatomines. A higher
percentage of metacyclic trypomastigotes for both parasite strains, Y and Berenice, was detected in the rectum extract of R. neglectus
in comparison to that from T. pseudomaculata. The same results were obtained with in vitro experiments, using parasites incubated
in urine from each of those vectors. The adhesion of parasites to the incubated rectum epithelial cells was also compared. In in-
cubations with the Y strain no significant differences were detected between the two triatomine species but, however, with the
Berenice strain the mean percentage of cells with adhered parasites was higher in R. neglectus than in T. pseudomaculata
Ó 2003 Elsevier Science (USA). All rights reserved.

Index Descriptors: Trypanosoma cruzi; developmental forms; metacyclogenesis; Rhodnius neglectus; Triatoma pseudomaculata

1. Introduction form of the parasite that is released in feces and urine.

ChagasÕ disease is a major public health problem in
Latin America. Carlos Chagas (1909) who first de-
scribed the disease in humans, identified the etiological
agent (Trypanosoma cruzi), its life cycle, the insect vec-
tor, and the wild mammalian species that functions as
host. The biological cycle of the parasite in the inver-
tebrate host was described in detail by Dias (1934),
showing the transformation of blood trypomastigotes
into the epimastigotes stages and the subsequent differ-
entiation into metacyclic trypomastigotes, the infectious
q to the cuticle of the rectum epithelium (Kleffman et al.,
This work is dedicated to the late Dr. Alina Perlowagora-
Szumlewicz.
*
Corresponding author. Fax: +55-21-2590-3495.
E-mail address: cbrasil@vm.uff.br (C.B. Mello).
Additional details complemented the characterization of
different forms of the parasite present along the digestive
tract of the vectors (see Alvarenga and Brener, 1978;
Brener, 1972; Garcia and Azambuja, 1991; Hoare, 1966;
Kollien and Schaub, 2000; Perlowagora-Szumlewicz and
Moreira, 1994; Schaub, 1989).
Epimastigotes forms replicate along the full length of
the digestive tract and in the Malpighian tubes, but the
production of metacyclic trypomastigotes occurs pre-
dominantly in the rectum (B€ oker and Schaub, 1984;
Schaub, 1989; Schaub et al., 1989). Metacyclogenesis
has been basically correlated to attachment of parasites
1998; Schmidt et al., 1998), parasite nutritional stress
(Camargo, 1964; Castellani et al., 1967; Goldenberg
et al., 1987), factors present in the rectum (Fraidenraich

0014-4894/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved.
doi:10.1016/S0014-4894(03)00072-9
 C.J. Carvalho-Moreira et al. / Experimental Parasitology 103 (2003) 102–111
et al., 1993; Garcia et al., 1995; Isola et al., 1981, 1986;
Schaub and L€ osch, 1988), and urine of the insect vector
(Kleffmann et al., 2000; Kollien and Schaub, 1997;
Rangel-Aldao et al., 1987).
There are more than 100 species of Triatominae in
the New World, but only some of them are important
vectors of ChagasÕ disease (revised by Garcia and Aza-
mbuja, 1991; Kollien and Schaub, 2000). The strain of
T. cruzi, vector species, and the bugÕs instar, can deter-
mine the degree of multiplication and differentiation of
the parasite inside it (Garcia and Dvorak, 1982; Garcia
et al., 1984; Gonzalez and Garcia, 1992; Mello et al.,
1996; Perlowagora-Szumlewicz and Moreira, 1994;
Perlowagora-Szumlewicz et al., 1990; Schaub, 1989).
In the present work, we investigated metacyclogenesis
in vivo and in vitro and compared the results in order to
identify the main mechanisms involved in the develop-
ment of T. cruzi in two different vectors, Rhodnius neg-
lectus and Triatoma pseudomaculata. In addition, T. cruzi
epimastigotes were incubated in urine and in extracts
from different compartments of the digestive tube of
these triatomines so that the production of metacyclic
trypomastigotes could be analyzed. Complementarily,
the epimastigotes were also incubated with vector rectum
tissue to quantify the parasite adhesion to epithelial cells.
2. Materials and methods
The experiments carried out involved two different
strains of T. cruzi isolated from human cases, the Y and
the Berenice strains. The Y strain, isolated in S~
ao Paulo,
Brazil, in 1950, was obtained from Professor Z. Brener
(FIOCRUZ-MG, Brazil) and the Berenice strain, iso-
lated in 1962, was donated by Dr. M.A. de Sousa (FI-
OCRUZ-RJ, Brazil). These strains were maintained and
grown at 28 °C in LIT media (Chiari and Camargo,
1984) according to Perlowagora-Szumlewicz and
Moreira (1994) and Mello et al. (1995).
Thirty guinea pigs weighing from 300 to 350 g were
inoculated with 0.2–0.3 ml of infected mouse blood
containing 1.0
105 parasites/ml. All guinea pigs devel-
oped parasitemia, which peaked within a period of 20–
27 days of inoculation with the Berenice strain and
within 13–27 days of inoculation with the Y strain.
Two species of Triatominae were used: T. pseudo-
maculata and R. neglectus. The insects were fed on live
chicken and raised as described by Perlowagora-Szum-
lewicz and Moreira (1994). Insects which had been
starved since their last meal, 30 days before, were in-
fected as fourth instar nymphs by feeding them on in-
fected guinea pigs that had been inoculated 27 days
previously. The blood meal contained an average of
3.3
105 and 2,8
105 parasites/ml of the Berenice and
Y strain of T. cruzi, respectively. Only fully engorged
bugs were used.
103
The entire gut tracts of the triatomines were removed
and homogenized (Mello et al., 1996) in 0.5 ml of a
phosphate-buffered saline (pH 7.2) at every 30 days period
and the insects having been fed more than a week before.
Parasite incubation was tested with the extract of
different compartments of the digestive tracts of T.
pseudomaculata and R. neglectus. In these experiments
the stomachs (anterior midgut or crop), intestines (small
intestine or posterior midgut), and rectum (hindgut) of
10 fifth instar larvae of each species were removed two
weeks after the engorgement. These were then pooled
and homogenized using a pellet pestle, respectively, in
1.0, 0.5, and 0.25 ml of a phosphate-buffered saline
(PBS, pH 7.2, 380 m/mosm) and centrifuged at 7000g for
10 min (Mello et al., 1996). The supernatants were
sterilized by filtering in Millex filters (0.20 lm). The in-
cubations in digestive tube extracts were set up with
40 ll of each homogenate and 10 ll of cultures washed
three times containing 3.0
107 parasites/ml of epim-
astigotes in the growth phase from the Berenice and Y
strains. Parasite development was followed-up on days
2, 7, 14, and 21. Ten samples per group were drawn but
only six were counted per day. The four additional
samples were drawn to allow for possible contamination
and, therefore, serve as replacements.
Washed epimastigote cultures from the Y and Bere-
nice strains, obtained as already described and adjusted
for 3.0
107 /ml, were incubated in urine taken from
different triatomine species, which were sterilized by
filtering through Millex filters (0.20 lm). The parasite
suspensions were incubated in 40 ll of R. neglectus and
T. pseudomaculata urine, collected during a 24-h period
after feeding (Garcia et al., 1989). The numbers and
developmental stage of the parasites were analyzed in
Neubauer hemocytometers, after the first and the second
days of incubation.
The assays on adhesion percentages to rectum epi-
thelium were made from fragments of rectal ampullae of
R. neglectus and T. pseudomaculata that were gently
dissected in PBS. Washed epimastigotes (1.5–5
106 /
ml) were incubated for 30 min with the insect tissues.
For observation of parasite adhesion these fragments
were placed on slides with the coverslips and the number
of adhered parasites was counted by using a phase
contrast microscopy (400
). Five slides were analyzed
per group, 20 cells being counted in two different fields
per slide (200 cells per group). Percentage of cells with
parasite adhesion as well as the number of parasites
attached per cell was then estimated.
For the in vivo and in vitro assays, 10 ll from each of
six samples was examined in a counting chamber
(Neubauer hemocytometer). The classification of the
distinct morphological forms (tripomastigote and epi-
mastigote) was made based on cell shape, size, width,
and motility. While the tripomastigote exhibits the
characteristic movement of the undulating membrane,
104 C.J. Carvalho-Moreira et al. / Experimental Parasitology 103 (2003) 102–111

present along its whole body length, the epimastigote

forms show a rotating movement only in the flagellum.
The other intermediary developmental forms were
classified in stained smears (showed in percentages). In
the fresh material the parasites were classified as epim-
astigotes, trypomastigotes, and other forms. In the fixed
and stained slides they were called transitional forms,
(i.e.) the intermediary stages between epimastigotes and
metacyclic tripomastigotes.
At least five sometimes six smears were made from
each sample, which were then stained according to the
May–Grunwald–Giemsa method (Perlowagora-Szumle-
wicz and Moreira, 1994). One hundred flagellates were
analyzed per smear and classified according to Schaub
(1989) and Perlowagora-Szumlewicz and Moreira (1994).
The protein concentration observed in each of these
distinct extracts from different parts of the digestive
tract was quantified through the use of the Protein
Dosage Kit (Bio-Rad Company). Bovine serum albumin
(BSA) was used as standard (Lowry et al., 1951).
Statistical analyses were made using the non-para-
metric test of Mann–Whitney (Statistics for Windows-
Release 4.2, Stat Soft Inc., 1993).
3. Results
In vivo experiments produced a low percentage of
metacyclic forms in all analyzed insects on the 30th day
after the infective meal, as shown in Fig. 1. In the di-
gestive tubes of R. neglectus 4.47 and 6.35% of meta-
cyclic forms were found but in T. pseudomaculata, 0.77
and 0.75%, respectively, relative to infections with Y and
Berenice strains. From the 60th day after infection, the
percentage of metacyclic trypomastigote revealed a
tendency to increase. The biggest difference between the
two species of Triatominae studied appeared on the
120th and 180th days after infective feeding. After these
periods of time had elapsed the parasite count in the gut
extracts showed concentrations of 67.03%; 72.34% of
metacyclic forms for R. neglectus and 6.84%; 2.36% for
T. pseudomaculata, respectively, for infections with the
Y and Berenice strains (Fig. 1).
In Graphs A–D from Figs. 2–4, bars represent the
total numbers (left-side) and lines, the percentages
(right-side) of developmental stages of parasites incu-
bated in gut homogenates.
3.1. Incubations with stomach extracts
In the incubations using stomach extracts (Fig. 2)
from R. neglectus (A and B) and T. pseudomaculata (C
and D) we observed, in general, a great predominance
and maintenance of high percentages of epimastigote
forms in the four examined periods after incubation, that
is, on the 2nd, 7th, 14th, and 21st day. On the last day of
Fig. 1. Percentage of metacyclic forms in different vector species in-
fected with the Y and Berenice strains of Trypanosoma cruzi. The in-
sects were examined at different times post-infection after a single meal
on infected guinea pigs. At least five sometimes six bugs were examined
per species and the examination was made by using the entire digestive
tract—where all the forms could be found, regardless of their devel-
opment sites, ground in physiological saline. Smears were confectioned
(n ¼ 6) and stained by May–Grunwald–Giemsa method. (–r– R.
neglectus; –N– T. pseudomaculata.)
incubation the lowest percentages of metacyclic forms
observed were 3.8% (2.2
105 forms) and 7.4%
(1.6
105 forms), respectively, in the incubations of the
Y and Berenice strains of T. cruzi in extracts of T.
pseudomaculata. While the highest percentages registered
were 12.4% (5.2
105 forms) and 14.8% (7.2
105
forms), with the incubations in extracts from R. neglectus
inoculated, respectively, with the same above-mentioned
strains. The transitional forms between epimastigote and
metacyclic started to appear on the second day of incu-
bation and in low values. The average of 19.0% (3.9
105
forms) was observed on the 21st day after the incubation
(dai), using extracts from T. pseudomaculata inoculated
with the Berenice strain. Thus, on the 21st day, a high
mortality rate was observed to be affecting mainly the
epimastigotes, reducing total parasite number, and in-
creasing, therefore, the proportion of transitional forms.
With incubations in stomach extracts of R. neglectus, a
progressive growth of metacyclic forms up to the 21st dai
was observed. The stomach extracts showed a protein
concentration of 234.66 lg/ml in R. neglectus and of
262.19 lg/ml in T. pseudomaculata.
3.2. Incubations with small intestine extracts
The incubations in small intestine extracts (Fig. 3) also
showed a great prevalence of epimastigotes. Percentages
varied from 46.4% (11.7
105 forms) with incubations in
 C.J. Carvalho-Moreira et al. / Experimental Parasitology 103 (2003) 102–111 105

Fig. 2. Incubation of crop extracts from Rhodnius neglectus (A and B) and Triatoma pseudomaculata (C and D) with the Y strain (A and C) and the
Berenice strain (B and D) of Trypanosoma cruzi epimastigotes from culture. The left axis of ordinates quantifies the columns of the graphics, in-
dicating the number of parasites counted in the Neubauer chamber. The right axis of ordinates shows on the lines the percentage of each kind of
counted parasite in fixed and stained slides. The SD (n ¼ 6) are indicated by the bars in each column or line point ( epimastigotes; other
forms; metacyclics, –N– percentage of epimastigotes; –d– percentage of transitional forms; and –}– percentage of metacyclic forms).

Fig. 3. Incubation of intestine extracts from Rhodnius neglectus (A and B) and Triatoma pseudomaculata (C and D) with the Y strain (A and C) and
the Berenice strain (B and D) of Trypanosoma cruzi epimastigotes from culture. The left axis of ordinates quantifies the columns of the graphics,
indicating the number of parasites counted in the Neubauer chamber. The right axis of ordinates shows on the lines the percentage of each kind of
counted parasite in fixed and stained slides. The SD (n ¼ 6) are indicated by the bars in each column or line point ( epimastigotes; other
forms; metacyclics, –N– percentage of epimastigotes; –d– percentage of transitional forms; and –}– percentage of metacyclic forms.
106 C.J. Carvalho-Moreira et al. / Experimental Parasitology 103 (2003) 102–111

Fig. 4. Incubation of rectum extracts from Rhodnius neglectus (A and B) and Triatoma pseudomaculata (C and D) with the Y strain (A and C) and the
Berenice strain (B and D) of Trypanosoma cruzi epimastigotes from culture. The left axis of ordinates quantifies the columns of the graphics, in-
dicating the number of parasites counted in the Neubauer chamber. The right axis of ordinates shows on the lines the percentage of each kind of
counted parasite in fixed and stained slides. The SD (n ¼ 6) are indicated by the bars in each column or line point ( epimastigotes; other
forms; metacyclics, –N– percentage of epimastigotes; –d– percentage of transitional forms; and –}– percentage of metacyclic forms).

extracts from R. neglectus (21 dai), right up to 84.8%
(42.6
105 forms) with incubations in extracts from
T. pseudomaculata, both using the Berenice strain (14
dai). Very few metacyclic forms were found with the
incubations in small intestine homogenates from
T. pseudomaculata (1–3.8%). However, in the assays with
R. neglectus extracts, 10.4 and 13.4% of metacyclic try-
pomastigotes were detected after 21 days of incubations
with Y and Berenice, respectively. With incubations in
R. neglectus extract versus T. cruzi Y the column relative
to the mean number of metacyclic forms is absent be-
cause in this case, no metacyclic trypomastigotes were in
the analysis using the Neubauer chamber. Later, based
on kinetoplast position, stained smears allowed the re-
classification of various forms initially classified as
epimastigotes, as metacyclic forms, including many
‘‘other forms’’ among which are round forms.
In all intervals of days analyzed, the assays with small
intestine homogenates from R. neglectus, incubated with
Berenice strain of T. cruzi, showed a higher percentage of
metacyclic forms. In this group, a percentage of transi-
tional forms ranging between 19 and 31.4% was verified,
from the second day to the 21st day, the last day analyzed.
The small intestine extracts from R. neglectus and T.
pseudomaculata revealed a protein concentration of
42.52 and 39.97 lg/ml, respectively.
3.3. Incubations with rectum extracts
The counting of parasites in rectum extract incuba-
tions are summarized in Figs. 4(A)–(D). In all groups of
rectum incubations, a slow reductions of average num-
bers of epimastigote forms (absolute value and percen-
tile) were observed. Exceptions to this were the
incubations with rectum extracts of T. pseudomaculata
using the Y strain which suffered an abrupt fall on the
14th dai, due mainly to the mortality rates observed.
The number of transitional forms in rectum extracts of
T. pseudomaculata with Berenice strain ranged from
14.2% (2.7
105 forms), on the 7th dai, to almost 40.0%
(3.2
105 forms), on the 21st dai. In the same species,
the incubations with rectum homogenates using the Y
strain varied from 5.6% (1.5
105 ), on the 7th dai, to
19.6% (2.8
105 forms), on the 14th dai. The averages
observed with the Y strain in extracts from R. neglectus
varied from 5.8% (6.0
104 forms) on the 2nd dai to
29.2% (5.9
105 forms), on the 21st dai. Using the
Berenice strain, higher results were observed, ranging
from 7.0% (3.7
105 forms) on the 14th dai to 38%
(19.0
105 forms), on the 21st dai. Incubations in R.
neglectus extract produced increasing percentages of
metacyclic forms. The percentages of metacyclic try-
pomastigotes were 23.4 (5.4
105 forms) and 19.2%
 C.J. Carvalho-Moreira et al. / Experimental Parasitology 103 (2003) 102–111 107

Fig. 5. Incubation of Y (A) and Berenice (B) strains of Trypanosoma cruzi epimastigotes from culture with saline solution. The left axis of ordinate
quantifies the columns of the graphics, indicating the number of parasites counted in Neubauer chamber. The right axis of ordinates shows on the
lines the percentage of each kind of counted parasite in fixed and stained slides. The SD (n ¼ 6) are indicated by the bars in each column or line point
( epimastigotes; other forms; metacyclics, –N– percentage of eimastigotes; –d– percentage of transitional forms; –}– percentage of
metacyclic forms).

(19.0
105 forms) for the Y and Berenice strains, re-
spectively, on the 21st dai (Figs. 4A and B). The incu-
bations in T. pseudomaculata rectum extracts were led to
only a few numbers of metacyclic trypomastigotes,
ranging from 2.0 to 3.4% (Figs. 4C and D). Percent wise
the production of metacyclic trypomastigotes in rectum
extracts was significantly higher (p < 0:01) when results
from R. neglectus were compared to those from T.
pseudomaculata. However, no significant difference was
detected when the influence of parasite strains was
compared.
The protein concentration in rectum extracts was
16.99 and 6.38 lg/ml for R. neglectus and T. pseudo-
maculata, respectively. To avoid the influence of protein
concentration, the homogenates were prepared with the
same amounts of segments from digestive tubes, for
both vector species.
The difference between the protein concentrations of
the rectum homogenates was maintained, because pro-
tein amount alone could be a chief factor in metacy-
clogenesis.
transformation to metacyclic trypomastigotes was sig-
nificantly higher (p < 0:01) with incubations of epim-
astigotes in urine coming from R. neglectus than in that
coming from T. pseudomaculata, in both (Y and Bere-
nice) strains of T. cruzi (Figs. 6(A)–(D)). However, the
marked predominance of epimastigotes persisted until
the last day of incubation and always totaled above 80%
of counted parasites.
3.6. In vitro attachment
The parasites incubated in rectum epithelial cells
showed that epimastigotes from the Berenice strain ad-
hered to 56.0% of R. neglectusÕ epithelial cells against an
adhesion of 33.0% in the case of T. pseudomaculata
(p < 0:01). However, no such difference was detected
with the Y strain (Table 1). A rate not superior to 1.42,
indicating adhered parasites per cell attached in rectum
tissues from R. neglectus or T. pseudomaculata, was
observed (Table 1).

3.4. Incubation with saline solution (control) 4. Discussion

The incubations in saline solution (Fig. 5) showed a
high mortality rate of parasites and also an almost total
absence of metacyclic trypomastigotes during all ob-
served days. An increase in transitional forms was de-
tected from the second to the 7th dai. Apparently, these
parasites did not get to complete the transformation to
the metacyclic form.
3.5. Incubation with urine
Although incubations were run for only two days, the
results of the assays in vitro with urine collected from
R. neglectus and T. pseudomaculata corroborated the
observations of in vivo experiments. The percent
Perlowagora-Szumlewicz and Moreira (1994) com-
pared metacyclogenesis percentages in vivo among nine
different species of Triatominae infected with strain Y
T. cruzi. R. neglectus showed the highest percentage of
metacyclic trypomastigotes and T. pseudomaculata
showed the lowest. The present paper confirms that
R. neglectus produces more metacyclic forms in infections
with the Y strain, as well as exhibits the same difference
between these two vector species, when infected with the
Berenice strain of T. cruzi. The parasite count was
achieved by considering the totality of the digestive tract
and not only the rectum, which is the main compartment
of occurrence of metacyclogenesis (Schaub, 1989).
Additionally, the highest parasite density in the whole
108 C.J. Carvalho-Moreira et al. / Experimental Parasitology 103 (2003) 102–111

Fig. 6. Incubation of urine from Rhodnius neglectus (A and B) and Triatoma pseudomaculata (C and D) with the Y strain (A and C) and the Berenice
strain (B and D) of Trypanosoma cruzi epimastigotes from culture. The left axis of ordinates quantifies the columns of the graphics, indicating the
number of parasites counted in the Neubauer chamber. The right axis of ordinates shows on the lines the percentage of each kind of counted parasite
in fixed and stained slides. The SD (n ¼ 6) are indicated by the bars in each column or line point ( transitional forms; metacyclics; –s–
percentage of transitional forms; –}– percentage of metacyclics).

Table 1
In vitro adhesion of epimastigotes from Berenice and Y strain of Trypanosoma cruzi in epithelial cells of rectum fragments from Rhodnius neglectus
and Triatoma pseudomaculata
Rhodnius neglectus Triatoma pseudomaculata
Y Berenice Y Berenice
a
Percentage of adhered cells 42.9 (16.39) 56.0 (0.40) 33.1 (12.52) 33.0 (1.20)
Number of attached parasites per adhered cell 1.10 1.42 1.11 1.32
a
Mean number (SD). n ¼ 200.

digestive tract was that of the rectal population after 20
days of incubation (Kollien and Schaub, 1998a). Fur-
thermore, this higher percentage of parasites in the rectum
had a tendency to increase during long periods of star-
vation (Kollien and Schaub, 1998b; Schaub and B€ oker,
1986; 2000) or often, upon refeeding (Schaub, 1989).
The studies carried out during the first 60 days, fo-
cusing on insects infected with the Y strain, always
showed a low percentage of metacyclics as well as
samples with few parasites. Our results with Y strain in
vivo are in agreement with Perlowagora-Szumlewicz and
Moreira (1994) who detected few or no metacyclic try-
pomastigotes in invertebrate hosts infected with the Y
strain of T. cruzi and mainly in insects analyzed during
the first 30 days after infection.
The experiments performed in vitro aimed at identi-
fying some of the mechanisms involved in the different
percentages of metacyclic forms of T. cruzi when they
are developing in R. neglectus or T. pseudomaculata.
This difference is related mainly to factors present in the
digestive tube environment (Garcia and Azambuja,
1991; Kollien and Schaub, 2000).
Incubations also used epimastigotes from culture in
PBS to control the effect of this buffer on the in vitro
transformation of the parasites. In these incubations, no
metacyclogenesis was detected for both Y and Berenice
strains of T. cruzi and many parasites died after a few
days. OÕDaly (1975) reported the differentiation of
metacyclic forms induced by nutritional stress in a poor
medium, but it was necessary to add fetal bovine serum
albumin as a stimulus. This result could explain the
appearance of some metacyclic trypomastigotes, in in-
cubations in extracts from the stomach and intestine,
because albumin from the blood meal is present in gut
 C.J. Carvalho-Moreira et al. / Experimental Parasitology 103 (2003) 102–111
contents. Additionally, these in vitro results could ex-
plain experiments that detected metacyclic trypom-
astigotes in homogenates from stomach of infected
triatomines (Cortez et al., 2002).
In all analyzed groups from insect digestive tube
homogenates extracts obtained from R. neglectus always
showed higher percentages of metacyclic trypomastig-
otes, in comparison with extracts from T. pseudomacu-
lata. Furthermore, when comparing the analysis of
homogenates obtained from each one of the three gut
compartments, a big difference in the percentage of
metacyclogenesis between the two species of Triatomi-
nae was detected in the rectum extracts. These results
are in accordance with those of in vitro (Fraidenraich
et al., 1993) and in vivo (Garcia et al., 1995) experi-
ments, which used a peptide derived from the hemo-
globin digestion that is present in the rectum of
Triatoma infestans. This peptide is related to the epi-
mastigote adenyl cyclase activation and the consequent
stimulus for metacyclogenesis (Flawi
a et al., 1997). The
higher efficiency of rectum homogenate in promoting
the metacyclogenesis could be related to these peptides
derived from the insectÕs digestive process. This differ-
ence among gut extracts could be enhanced if the anal-
ysis of the transformations takes into account the lower
protein concentration in homogenates from the rectum.
On the other hand, the apparently normal development
of T. cruzi was reported in the triatominae in absence of
blood (Alvarenga and Brener, 1978). However, Garcia
et al. (1995) demonstrated that just after a long starva-
tion period, the contents of the gut originating from
the last blood meal did not exert any influence on the
metacyclogenesis of T. cruzi, in the digestive tube of
R. prolixus.
In vitro experiments demonstrated that GraceÕs me-
dium, modified through addition of midgut extract,
which is rich in the peritrophic membrane from T. in-
festans, could increase differentiation from the epimas-
tigote to the metacyclic stage (Burgos et al., 1989). The
same work showed that metacyclogenesis percentage in
GraceÕs medium is never higher than 6% after 10 days of
incubation. However, with addition of gut homogen-
ates, this transformation reached approximately 56% of
the population. In the present paper a lower percentage
of metacyclogenesis was obtained, in comparison with
other works in which the incubation with gut extracts
was made in GraceÕs medium (Burgos et al., 1989; Isola
et al., 1981; Wood and Sousa, 1976). The influence of
the medium on the differentiation is still not elucidated.
In any case, in our experiments, we preferred to work
without the medium or the serum, in order to analyze
only the isolated effect of the digestive tube molecules.
On the other hand, the in vivo results are in accordance
with Contreras et al. (1985), who also had difficulties in
obtaining in vitro metacyclic forms from Y strain (4–
12%), in comparison with Dm28c clone (87%). They got
109
a higher percentage with the Y strain when incubating
the parasites from epimastigotes, which had been de-
rived from previously produced metacyclic trypom-
astigotes. The different degrees of the metacyclogenesis
detected between these two species of vectors would
probably be more conspicuous if the homogenates for
analysis had been obtained at different periods of time
after the blood feeding or if the epimastigotes had
originated from the vectors.
As a whole the results obtained here from incubation
in homogenates are in agreement with in vivo observa-
tions, when comparisons were made taking into account
only the percentage of metacyclic trypomastigotes, as
well as this same stage, together with the transitional
forms. The transitional developmental forms of the
parasite in the gut of the insect vector were found in
both experiments, in vivo (Dias, 1934; Perlowagora-
Szumlewicz and Moreira, 1994; Schaub, 1989) and in
vitro (Bonaldo et al., 1988; Gonzales-Perdomo et al.,
1988). These forms are intermediate stages found during
the migration of the kinetoplast from the anterior ex-
tremity of the parasite to the posterior one (or vice
versa). These stages are classified according to the ki-
netoplast position in relation to the nucleus and ac-
cording to the cell morphology and its flagellum
(Schaub, 1989).
Some specific genes can be activated during the
metacyclogenesis process (Krieger and Goldenberg,
1998) and at least one of them has its expression regu-
lated by cAMP (Heath et al., 1990). It has been sug-
gested that there is participation of the cAMP excreted
by the triatominesÕ urine in the transformation process
of epimastigotes into metacyclic forms of T. cruzi
(Garcia and Azambuja, 1991 and Kollien and Schaub,
2000). The in vitro metacyclogenesis has been success-
fully conducted in the vectorÕs mimetic urine medium
(Contreras et al., 1985, 1988) called TAU (triatomine
artificial urine).
Our experiments using urine were made with only
two days of incubation and showed, therefore, a low
percentage of transformation into epimastigotes. On the
other hand the quantity of trypomastigotes was higher
with incubations in urine of R. neglectus than that in
urine of T. pseudomaculata. This difference, as observed
with incubations with gut homogenates, was detected
for both the Y and the Berenice strains of T. cruzi. The
raising of T. pseudomaculata in our colony of triato-
mines was very difficult. Moreover, this vector excretes a
small volume of urine, at least for the first days fol-
lowing blood feeding (personal observation). These
characteristics prevented the experiments with urine in-
cubation to be carried out over long periods.
The chemical composition of the urine of the triato-
mine showed small variations between samples collected
at different periods of time after the blood meal (Kollien
et al., 2001; Kollien et al., 1998). Preliminary results
110 C.J. Carvalho-Moreira et al. / Experimental Parasitology 103 (2003) 102–111
indicated that proteins found at specific moments of the
urine flow should induce the metacyclogenesis (Kleff-
mann et al., 2000). In addition, the efficiency of in vitro
transformation into metacyclic trypomastigotes could
be correlated with the adhesion of the parasites to the
epithelial cells of the vectorÕs rectum (Kleffmann et al.,
2000). The feeding induces a detachment of the try-
pomastigotes from the posterior rectal wall and, thus,
the population of parasites that will be released in the
urine is composed totally of metacyclic trypomastigotes
(Schaub and L€ osch, 1988).
The attachment of T cruzi to the gut epithelium of the
triatomine involves a hydrophobic interaction (Schmidt
et al., 1998). Some results indicate that only the at-
tachment of the epimastigotes does not induce the
metacyclogenesis, but can enhance the efficiency of this
transformation (Bonaldo et al., 1988; Kleffman et al.,
1998). The epimastigotes forms of the Berenice strain T.
cruzi showed a better capacity to adhere to the rectum
epithelial cells of R. neglectus than to the same cells of T.
pseudomaculataÕs tissue. However, the number of para-
sites attached per cell was practically the same in the
incubation tests carried out with both species of triato-
mine, R. neglectus and T. pseudomaculata. The attach-
ment experiments conducted with the Y strain of T.
cruzi did not reveal any difference between the two
species of triatomine.
Incubations with urine of R. neglectus showed early
metacyclogenesis if compared to experiments using ex-
tracts from the gut. All groups analyzed in vitro, with
material obtained from R. neglectus, showed a higher
efficiency of metacyclogenesis than the groups that used
material from T. pseudomaculata. However, in all incu-
bations either with gut homogenates or urine samples, a
lower percentage of metacyclic trypomastigotes was
observed as compared to the in vivo results. Conversely,
the higher population of metacyclic forms started being
detected in vivo only after a long period of infection, as
discussed above. Additionally, we believe that the small
differences verified with the in vitro experiments, when
added up, could also explain the higher in vivo difference
as related to the capacity of R. neglectus and T.
pseudomaculata to produce metacyclic trypomastigotes.
Apparently, the development of T. cruzi in the inver-
tebrate host is also likely to be modulated by a number of
factors that are strongly connected to each moment of the
inter feeding periods. The results in this paper support the
hypothesis that the regulation of the metacyclogenesis at
least depends on the physiological stage and on the in-
trinsic characteristics of each vector species.
Acknowledgments
This work was supported by grants from Conselho
Nacional de Desenvolvimento Tecnol
ogico, PADCT,
Fundacß~ao Oswaldo Cruz, FAPERJ and IOC/FIO-
CRUZ. CBM, MSG, ESG, and PA are CNPq research
fellows.
References
Alvarenga, N.J., Brener, Z., 1978. Development of Trypanosoma cruzi
in the absence of blood. Acta Tropica 35, 315–317.
B€
oker, C.A., Schaub, G.A., 1984. Scanning electron microscopic
studies of Trypanosoma cruzi in the rectum of this vector Triatoma
infestans. Zietschrift f€
ur Parasitenkunde 70, 459–469.
Bonaldo, M., Souto-Padron, T., De Souza, W., Goldenberg, S., 1988.
Cell–substrate adhesion during Trypanosoma cruzi differentiation.
Journal of Cellular Biology 106, 1349–1358.
Brener, Z., 1972. A new aspect of Trypanosoma cruzi life cycle in the
invertebrate host. Journal of Protozoology 19, 23–27.
Burgos, M.H., Gutierrez, L.S., Lammel, E., Isola, E.L.D., 1989.
Midgut extract rich in peritrophic membrane from Triatoma
infestans induces differentiation of Trypanosoma cruzi. Microscopia
Electronica y Biologia Celular 13, 151–166.
Camargo, E.P., 1964. Growth and differentiation of Trypanosoma
cruzi. I—Origin of metacyclic trypanosomes in liquid media.
Revista do Instituto de Medicina Tropical de S~ao Paulo 6, 93–100.
Castellani, O., Ribeiro, L.V., Fernandes, J.F., 1967. Differentiation of
Trypanosoma cruzi in culture. Journal of Protozoology 14, 447–
451.
Chagas, C., 1909. Nova tripanosom
ıase humana. Estudos sobre a
morfologia e o ciclo evolutivo do Schizotrypanum cruzi n. gen., n.
sp., agente etiol
ogico de nova entidade m
orbida do homem.
Mem
orias do Instituto Oswaldo Cruz 1, 159–218.
Chiari, E., Camargo, E.P., 1984. Culture and cloning of Trypanosoma
cruzi. In: Morel, C.M. (Ed.), Genes and Antigens of Parasites, A
Laboratory Manual. Fundacß~ao Oswaldo Cruz, World Health
Organization, Rio de Janeiro, pp. 23–26.
Contreras, V.T., Sales, J.M., Thomas, N., Morel, C.M., Goldenberg,
S., 1985. In vitro differentiation of Trypanosoma cruzi under
chemically defined conditions. Molecular and Biochemical Parasi-
tology 16, 315–327.
Contreras, V.T., Araujo-Jorge, T.C., Bonaldo, M.C., Thomaz, N.,
Barbosa, H.S., Meirelles, M.N.S.L., Goldenberg, S., 1988. Biolog-
ical aspects of the Dm 28c clone of Trypanosoma cruzi after
metacyclogenesis in chemically defined media. Mem
orias Instituto
do Oswaldo Cruz 83, 123–133.
Cortez, M.G.R., Gonzalez, M.S., Cabral, M.M.O., Garcia, E.S.,
Azambuja, P., 2002. Dynamic development of Trypanosoma cruzi
in Rhodnius prolixus: role of decaptation and ecdysone therapy.
Parasitology Research 88, 697–703.
Dias, E., 1934. Estudos sobre o Schyzotrypanum cruzi. Mem
orias do
Instituto Oswaldo Cruz 28, 1–110.
Flawi
a, M.M., T
ellez-I~no
n, M.T., Torres, H.N., 1997. Signal trans-
duction mechanisms in Trypanosoma cruzi. Parasitology Today 13,
30–33.
Fraidenraich, D., Pe~na, C., Isola, E.L., Lammel, E.M., Coso, O., A~ nel,
A., Pongor, S., Baralle, F., Torres, H.N., Flawi
a, M.M., 1993.
Stimulation of Trypanosoma cruzi adenyl cyclase by an a-globin
fragments from Triatoma hindgut: effect of differentiation of
epimastigote to trypomastigote forms. Proceedings of National
Academy of the Sciences, USA 90, 10140–10144.
Garcia, E.S., Azambuja, P., 1991. Development and interactions of
Trypanosoma cruzi within the insect vector. Parasitology Today 7,
240–244.
Garcia, E.S., Dvorak, J.A., 1982. Growth and development of two
Trypanosoma cruzi clones in the insect Dipetalogaster maximus.
American Journal of Tropical Medicine and Hygiene 31, 359–362.
 C.J. Carvalho-Moreira et al. / Experimental Parasitology 103 (2003) 102–111
Garcia, E.S., Gonzalez, M.S., Azambuja, P., Baralle, F.E., Fraidenra-
ich, D., Torres, H.N., Flawia, M.M., 1995. Induction of Trypan-
osoma cruzi metacyclogenesis in the gut of the hematophagous
insect vector, Rhodnius prolixus, by hemoglobin and peptides
carrying ad -globin sequences. Experimental Parasitology 81, 255–
261.
Garcia, E.S., Subrahmanyam, B., Muller, Th., Rembold, H., 1989.
Absorption and storage, organ distribution and excretion if dietary
(22, 23-3 H2 ) dihydro-azadirachtin A in the blood-feeding bug,
Rhodnius prolixus. Journal of Insect Physiology 35, 742–749.
Garcia, E.S., Vieira, E., Lima Gomes, J.E.P., Goncßalves, A., 1984.
Molecular biology of the interaction Trypanosoma cruzi/inverte-
brate host. Mem
orias do Instituto Oswaldo Cruz 79 (Suppl), 33–
37.
Goldenberg, S., Contreras, V.T., Bonaldo, M.C., Salles, J.M., Lima
Franco, M.P.A., Lafaille, J.J., Gonzales-Perdomo, M., Linss, J.,
Morel, C.M., 1987. In vitro differentiation systems for the study of
differential gene expression during Trypanosoma cruzi develop-
ment. In: Agabian, N., Goodman, H., Nogueira, N. (Eds.),
Molecular Strategies of Parasitic Invasion. UCLA Symposyum.
A.R. Liss, New York, pp. 203–212.
Gonzales-Perdomo, M., Romero, P., Goldenberg, S., 1988. Cyclic
AMP and adenylate cyclase activators stimulate Trypanosoma cruzi
differentiation. Experimental Parasitology 66, 205–212.
Gonzalez, M.S., Garcia, E.S., 1992. Effect of azadirachtin on the
development of Trypanosoma cruzi in different triatominae insect
vectors: long term and comparative studies. Journal of Invertebrate
Pathology 60, 201–205.
Heath, S., Hieny, S., Sher, A., 1990. A cyclic-AMP inducible gene
expressed during the development of infective stages of Trypano-
soma cruzi. Molecular and Biochemical Parasitology 43, 133–142.
Hoare, C.A., Wallace, F.G., 1966. Developmental stages of trypano-
somatid flagellates: a new terminology. Nature 212, 1385–1386.
Isola, E.L.D., Lammel, E.M., Gonzalez Cappa, S.M., 1981. Influence
of organ extract of Triatoma infestans on differentiation of
Trypanosoma cruzi. Journal of Parasitology 67, 53–58.
Isola, E.L.D., Lammel, E.M., Gonzalez Cappa, S.M., 1986. Trypan-
osoma cruzi: differentiation after interaction of epimastigotes and
Triatoma infestans intestinal homogenate. Experimental Parasitol-
ogy 62, 329–335.
Kleffman, T., Schmidt, J., Schaub, G.A., 1998. Attachment of
Trypanosoma cruzi epimastigotes to hydrophobic substrates and
use of this property to separate stages and promote metacyclogen-
esis. Journal of Eukaryotic Microbiology 45, 548–555.
Kleffmann, T., Lieke, T., Meyer, H.E., Schaub, G.A., 2000. Influence
of urinary proteins on metacyclogenesis in Trypanosoma cruzi.
Mem
orias Instituto Oswaldo Cruz 95 (Suppl. II), 342.
Kollien, A.H., Grospietsch, T., Kleffmann, T., Zerbst-Boroffka, I.,
Schaub, G.A., 2001. Ionic composition of the rectal contents and
excreta of the reduviid bug Triatoma infestans. Journal of Insect
Physiology 47, 739–747.
Kollien, A.H., Meyer, H.E., Grospietsch, T., Zerbest-Boroffka, I.,
Kleffmann, T., Schaub G.A., 1998. Correlation of the development
of Trypanosoma cruzi with the physiology in the rectum of the
vector. In: IX International Congress of Parasitology. Makuhan
Messe, Chiba, Japan, pp. 667–670.
Kollien, A.H., Schaub, G.A., 1997. Trypanosoma cruzi in the rectum of
the bug Triatoma infestans: effects of blood ingestion of the vector
and artificial diuresis. Parasitology Research 83, 781–788.
111
Kollien, A.H., Schaub, G.A., 1998a. The development of Trypanosoma
cruzi (Trypanosomatidae) in the reduviid bug Triatoma infestans
(Insecta): influence of starvation. Journal of Eukariotic Microbi-
ology 45, 59–63.
Kollien, A.H., Schaub, G.A., 1998b. The development of Trypanosoma
cruzi (Trypanosomatidae) in the reduviid bug Triatoma infestans
(Insecta): influence of starvation. Journal of Eukaryotic Microbi-
ology 45, 59–63.
Kollien, A.H., Schaub, G.A., 2000. The development of Trypanosoma
cruzi in Triatominae. Parasitology Today 16, 381–387.
Krieger, M.A., Goldenberg, S., 1998. Representation of differential
expression: a new approach to study differential gene expression in
trypanosomatids. Parasitology Today 14, 163–166.
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951.
Protein measurement with the folin phenol reagent. Journal of
Biological Chemistry 193, 265–275.
Mello, C.B., Azambuja, P., Garcia, E.S., Ratcliffe, N.A., 1996.
Differential in vitro and in vivo behaviour of three strains of
Trypanosoma cruzi in the gut and hemolymph of Rhodnius prolixus.
Experimental Parasitology 82, 112–121.
Mello, C.B., Garcia, E.S., Ratcliffe, N.A., Azambuja, P., 1995.
Trypanosoma cruzi and Trypanosoma rangeli: interplay with
hemolymph components of Rhodnius prolixus. Journal of Inverte-
brate Pathology 65, 261–268.
OÕDaly, J., 1975. A new liquid medium for Trypanosoma (Schizotry-
panum) cruzi. Journal of Protozoology 22, 265–270.
Perlowagora-Szumlewicz, A., Moreira, C.J.C., 1994. In vivo differen-
tiation of Trypanosoma cruzi—1. Experimental evidence of the
influence of vector species on metacyclogenesis. Mem
orias Instituto
Oswaldo Cruz 89, 603–618.
Perlowagora-Szumlewicz, A., Muller, C.A., Moreira, C.J.C., 1990.
Studies in search of a suitable experimental insect model for
xenodiagnosis of hosts with ChagaÕs disease 4—The refleciton of
parasite stock in the responsiveness of different vector species to
chronic infection with different Trypanosoma cruzi stocks. Revista
de Sa
ude P
ublica 24, 165–177.
Rangel-Aldao, R., Triana, F., Fernandez, V., Comach, G., Abate, T.,
Montoreano, R., 1987. cAMP as an inducer of the cell differen-
tiation in Trypanosoma cruzi. Biochemistry International 17, 337–
344.
Schaub, G.A., B€ oker, C.A., 1986. Colonization of the rectum of
Triatoma infestans by Trypanosoma cruzi: influence of starvation
studied by scanning electron microscopy. Acta Tropica 43, 349–354.
Schaub, G.A., L€ osch, P., 1988. Trypanosoma cruzi: origin of metacyclic
trypomastigotes in the urine of the vector Triatoma infestans.
Experimental Parasitology 65, 174–186.
Schaub, G.A., 1989. Trypanosoma cruzi: quantitative studies of
development of two strains in small intestine and rectum of the
vector Triatoma infestans. Experimental Parasitology 68, 260–273.
Schaub, G.A., Gr€ unfelder, C.G., Simmermann, D., Peters, W., 1989.
Binding of lectin–gold conjugates by two Trypanosoma cruzi strains
in ampullae and rectum of Triatoma infestans. Acta Tropica 46,
291–301.
Schmidt, J., Kleffmann, T., Schaub, G.A., 1998. Hydrophobic attach-
ment of Trypanosoma cruzi to a superficial layer of the rectal cuticle
in the bug Triatoma infestans. Parasitology Research 84, 527–536.
Wood, D.E., Sousa, O.E., 1976. Trypanosoma cruzi: effects of Rhodnius
prolixus extracts on in vitro development. Revista do Instituto de
Medicina Tropical de S~ao Paulo 18, 93–96.