Experimental Parasitology 145 (2014) 118124

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Experimental Parasitology
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Rhodnius prolixus: Modulation of antioxidant defenses by Trypanosoma

rangeli
Daniela Cosentino-Gomes, Nathlia Rocco-Machado , Jos Roberto Meyer-Fernandes
Institute of Medical Biochemistry, Federal University of Rio de Janeiro (UFRJ), CCS, Bloco H, Cidade Universitria, Ilha do Fundo, 21941-590 Rio de Janeiro, RJ, Brazil
Institute of National Science and Technology of Structural Biology and Bioimage (INCTBEB), CCS, Bloco H, Cidade Universitria, Ilha do Fundo, 21941-590 Rio de Janeiro, RJ, Brazil

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 Infection with T. rangeli increases

oxidative stress in R. prolixus.

 T. rangeli promotes a decrease in

catalase and GPx activities in R.

prolixus.
 T. rangeli infection promotes an
increase in H2O2 level in R. prolixus
midgut.

a r t i c l e

i n f o

Article history:
Received 9 December 2013
Received in revised form 7 May 2014
Accepted 3 August 2014
Available online 14 August 2014
Keywords:
Trypanosoma rangeli
Rhodnius prolixus
Antioxidant enzymes
Reactive oxygen species
Parasitehost interaction

a b s t r a c t
Trypanosoma rangeli is a protozoan parasite of insects and mammals that is challenged by the constant
action of reactive oxygen species, generated either by its own metabolism or through the host immune
response. The aim of this work was to investigate whether T. rangeli is able to modify the redox state of its
insect vector, Rhodnius prolixus, through the modulation of such antioxidant enzymes as superoxide dismutase (SOD), catalase, and GPx present in the midgut of the insect. We veried that in R. prolixus fed
with blood infected with T. rangeli there is an increase in SOD activity in the anterior and posterior midguts. However, the activities of enzymes related to hydrogen peroxide and hydroperoxides metabolism,
such as catalase and GPx, were decreased in relation to the insect control group, which was only fed
blood. These changes in the redox state of the vector led to an increase in lipid peroxidation and thiol
oxidation levels in the anterior and posterior midgut tissues. We also veried that the addition of
1 mM GSH in the blood meal of the infected insects increased the proliferation of these parasites by
50%. These results suggest that there is an increase in oxidative stress in the insect gut during T. rangeli
infection, and this condition could contribute to the control of the proliferation of these parasites.
2014 Elsevier Inc. All rights reserved.

1. Introduction
Trypanosoma rangeli is a South American trypanosomatid that is
able to infect vertebrate and invertebrate hosts. However, in contrast to Trypanosoma cruzi, the etiologic agent of Chagas disease,
Abbreviations: SOD, superoxide dismutase; GPx, glutathione peroxidase; GSH,
reduced glutathione.
Corresponding authors at: Instituto de Bioqumica Mdica, Universidade
Federal do Rio de Janeiro, Av. Carlos Chagas Filho, 373, Ilha do Fundo, Rio de
Janeiro 21941-902, Brazil.
E-mail addresses: rocco@bioqmed.ufrj.br (N. Rocco-Machado), meyer@bioqmed.
ufrj.br (J.R. Meyer-Fernandes).
http://dx.doi.org/10.1016/j.exppara.2014.08.002
0014-4894/ 2014 Elsevier Inc. All rights reserved.

T. rangeli is considered unable to elicit pathology in mammals,

though it is detrimental to its insect vector (Cuba-Cuba, 1998;
Guhl and Vallejo, 2003). T. rangeli co-exists with T. cruzi in northern
South America, which poses some problems for diagnosis
(Martnez et al., 1993), mainly because of a high immunological
cross-reactivity between these two parasites (Basso et al., 2004;
Labriola and Cazzulo, 1995). The life cycle of T. rangeli in its vertebrate host is poorly known. In contrast, after ingestion as a trypomastigote, T. rangeli is known to multiply as an epimastigote in the
midgut of its invertebrate host, invade the hemolymph and hemocytes to continue growth, and complete development in the salivary gland, the site where metacyclogenesis occurs (Hoare, 1972).

D. Cosentino-Gomes et al. / Experimental Parasitology 145 (2014) 118124

Protozoan parasites are susceptible to a large number of oxidative conditions, including the reactive oxygen species (ROS) that
are part of the immune system of the insect vector. ROS can also
be produced during the breakdown of hemoglobin in the stomach
of the insect vector as a consequence of the release of large
amounts of heme or as a byproduct of the parasites own aerobic
metabolism (Finzi et al., 2004).
Rhodnius prolixus is a hematophagus insect and a potential
transmitter of many pathogens. Only insect species of the genus
Rhodnius present infective forms of T. rangeli in their salivary
glands, and these insects are highly susceptible to parasitemia
(Garcia et al., 2009). The establishment of T. rangeli infection in
the gut and in the hemocoel of its vector insect is possibly regulated by a variety of chemical and physiological processes. As the
intestine is the rst environment for the establishment of T. rangeli
infection, the many enzymes, proteins, and products of blood
digestion present in this environment may have fundamental roles
in the development of the parasite (Azambuja and Garcia, 2005;
Garcia et al., 2009).
In other insects, such as the wax moth Galleria mellonella,
increased ROS production and the consequent modulation of antioxidant enzymes has been described as an indirect mechanism of
the defense against pathogens (Dubovskiy et al., 2008; Wang
et al., 2001). Aedes aegypti infected with the bacteria Wolbachia also
shows an increase in the ROS levels. ROS up-regulation resulted in
activation of the Toll pathway, which mediated the antioxidant
defenses. This immune pathway is also responsible for activation
of antimicrobial peptides (Pan et al., 2011). Lee et al. (2013) shows
that Drosophila melanogaster infected with pathogenic bacteria
have an increase in the ROS levels due to the activation of dual oxidase (DUOX), a member of nicotinamide adenine dinucleotide
phosphate (NADPH) oxidase family. Symbiotic microbes have
lower ROS-producing effects. Molina-Cruz et al. (2008) show that
genetic differences in systemic hydrogen peroxide (H2O2) levels
between Anopheles gambiae strains have broad effects in their
immune response to Plasmodium and other pathogens such as bacteria. The higher the systemic ROS levels in a given strain, the better the mosquitoes survive a bacterial challenge. Dietary
supplementation with antioxidants (vitamin C or uric acid) can
dramatically reduce the mosquitos ability to mount an effective
antibacterial response. Anopheles albimanus infected with Plasmodium berghei produces nitric oxide and H2O2 in the midgut, that
function as signals for the activation the mosquito system immune
response (Herrera-Ortiz et al., 2010). Buarque et al. (2013) shows
that Triatoma infestans infected with T. cruzi exhibits a downregulation of the antioxidant enzyme thioredoxin reductase.
Our goal in this work is verify the changes on redox state of the
insect vector R. prolixus, including the modulation of antioxidant
enzymes such as superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx), as well as, the level of H2O2 in the midgut
of the insect promoted by the infection with T. rangeli.
2. Materials and methods
2.1. Materials
All reagents were purchased from Merck (Darmstadt, Germany)
or Sigma Chemical Co. (St. Louis, MO, USA). The water used in the
preparation of all solutions was ltered using a four-stage Milli-Q
system (Millipore Corp., Bedford, MA, USA).
2.2. Microorganisms
Epimastigote forms of T. rangeli strain H14 (supplied by Dr.
Maria Auxiliadora Sousa, from Coleo de Tripanossomatdeos,

119

Instituto Oswaldo Cruz, Rio de Janeiro, Brazil) were maintained

at 28 C in liver infusion tryptose (LIT) medium adjusted to pH
7.2 with HCl and supplemented with 20% fetal bovine serum. The
parasites were grown for 7 days in culture medium, collected by
centrifugation at 1500g at 4 C for 15 min, and washed three
times in a buffer solution containing 50 mM TrisHCl (pH 7.2),
100 mM sucrose, and 20 mM KCl.
2.3. R. prolixus and infection with T. rangeli
The insects were obtained from a colony of R. prolixus maintained at 28 C and relative humidity of 7080% under a photoperiod of 12 h light/12 h dark. Adult insects were fed live rabbit blood
at 3-week intervals, beginning 15 days after molting. Animal care
and experimental protocols were approved by the Committee for
Evaluation of Animal Use for Research of Federal University of
Rio de Janeiro (CAUAP-UFRJ), under the registry #IBQM001.
Male insects at the 5th stage and fasted for at least 4 weeks
were randomly selected and fed human blood containing citrate
via articial feeding. For infection, 5  106 cells/ml of T. rangeli
epimastigotes were collected in the stationary phase and added
to human blood previously inactivated for 1 h at 56 C, as previously described by Gonzalez et al., 2013. After an incubation period, the insects were dissected to remove portions of the anterior
and posterior midguts (Gomes et al., 2003).
2.4. Dissection of R. prolixus
After the insects were dissected, the anterior and posterior midguts were isolated and washed in phosphate-buffered saline (PBS),
pH 7.4, to remove all of the contents of the lumen, as described by
Grillo et al. (2007). The two intestinal segments were then homogenized in the same buffer containing a cocktail of protease inhibitors (Sigma) and 1 mM of phenylmethylsulfonyl chloride (PMSF).
We used 200 ll and 150 ll of the buffer to homogenize the anterior and posterior midguts, respectively. The samples were centrifuged at 11,000g for 5 min, and the supernatants were used for
the enzyme activity measurements. The total protein concentration was determined by the method of Lowry et al. (1951) using
bovine serum albumin as the standard.
2.5. Catalase assay
To measure catalase activity, 0.05 mg/ml of protein was added
to a reaction medium containing 100 mM TrisHCl (pH 7.4). The
reaction was initiated by the addition of the substrate, 8 mM
H2O2. The dismutation of H2O2 was monitored in a quartz cuvette
using a SpectraMax microplate reader (Molecular Devices, LLC,
USA) at a wavelength of 240 nm for 300 s, as described by Paes
et al. (2001). The catalase activity was calculated using the molar
extinction coefcient of H2O2, 36 M1  cm1 and is expressed in
mmol of H2O2 ptn  mg1  min1.
2.6. Glutathione peroxidase assay
To measure GPx activity, 0.05 mg/ml of protein was added to a
reaction medium containing PBS (pH 7.4), 10 mM sodium azide,
1 mM EDTA, 1 mM GSH, 0.1 mM NADPH, 2 U/ml glutathione
reductase; 0.25 mM H2O2 was used as the substrate. The sample
(10 lL) was pre-incubated in the medium, and the reaction was
initiated by the addition of hydrogen peroxide. The oxidation of
NADPH was monitored in a quartz cuvette using a SpectraMax
microplate reader (Molecular Devices, LLC, USA) at a wavelength
of 340 nm for 300 s; readings were obtained every 30 s, as
described by Paes and Oliveira (1999). The GPx activity was calculated by subtracting the absorbance of the blank (in the absence of

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D. Cosentino-Gomes et al. / Experimental Parasitology 145 (2014) 118124

sample) and using the molar extinction coefcient of NADPH,

6.22 mM1  cm1; the activity is expressed in nmol of NADPH
ptn1  mg1  min.
2.7. Superoxide dismutase assay
For measurement of the SOD activity, a 20-lL aliquot of the
sample was added to a reaction medium containing PBS,
0.16 mU/ml xanthine oxidase, 20 lM cytochrome c, 0.1 mM EDTA,
20 mM KCN; 0.1 mM xanthine was used as the substrate. The reaction was initiated by the addition of 20 lL of xanthine to the reaction medium in a 96-well plate. Readings were obtained following
the reduction of cytochrome c at 550 nm using a SpectraMax
microplate reader (Molecular Devices, LLC, USA), with 300 readings
every 30 s. This method uses the xanthine-xanthine oxidase system as a generator of superoxide radicals, which are used for the
oxidation of cytochrome c. The activity of superoxide dismutase
is given by the ability of this enzyme to compete with cytochrome
c for superoxide. Thus, one unit of SOD activity corresponds to a
50% inhibition of the reaction between superoxide and cytochrome
c (Mccord and Fridovich, 1969; Kabil et al., 2007).
2.8. Hydrogen peroxide production
To measure the H2O2 production, we used homogenates of the
anterior (AM) and posterior midgut (PM) of 5 insects under each
condition from 3rd and 5th days. The homogenates were kept on
ice throughout the procedure. After maceration, supernatant samples 0.05 mg protein/ml were incubated for 20 min in a medium
containing PBS (pH 7.4), 1.7 lM Amplex Red and 6.7 U/ml horseradish peroxidase (HPR) in a nal volume of 200 ll (Meyer et al.,
2006). HRP catalyzes the reaction between Amplex Red and H2O2
forming the uorophore resorun, which was monitored at excitation and emission wavelengths of 563 (slit 5 nm) and 587 nm (slit
5 nm), respectively. After 20 min of reaction, H2O2 production was
then determined using a standard curve with known quantities of
H2O2.
2.9. Lipid peroxidation
The thiobarbituric acid method (TBARS), as described by Vynce
(1970), was utilized to measure lipid peroxidation. We used
homogenates of the anterior (AM) and posterior midgut (PM) of
8 insects under each condition. The homogenates were kept on
ice throughout the procedure. After maceration, the tissue samples
were incubated for 15 min in 10% trichloroacetic acid at a ratio of
2:1 acid:sample. The tubes were centrifuged at 2200g for 15 min
at 4 C, and 400 ll of the supernatant was removed and added to
an equal volume of 0.67% thiobarbituric acid (TBA). The samples
were incubated for 15 min at 100 C, and 200 ll of each sample
was placed in a well of 96-well plates and measured at 532 nm
using a SpectraMax microplate reader (Molecular Devices, LLC,
USA). The levels of lipid peroxidation were calculated from the
quantication of the malondialdehyde (MDA) formed by means
of a standard curve using known concentrations of 1,1,3,3-tetramethoxypropane (Sigma) and normalized to the protein concentration of each sample.
2.10. Quantication of oxidized thiol groups
The method based on the oxidation of RSH by 5,50 -dito-bis(2nitrobenzoic acid), also known as the Ellman reagent (DNTB),
was utilized for the measurement of oxidized (RSSR) and reduced
(RSH) thiol groups (Dubovskiy et al., 2008). A 50-lL sample of
AM and PM homogenates were incubated with 1 mM hydrochloric
acid for 20 min to form RSH. The pH of the reaction was neutralized

with NaOH (pH 7.0), and 50 ll of the original sample and 50 ll of

the decomposed sample were incubated with 500 ll of 0.1% DTNB
in PBS for 10 min at 37 C. The absorbance of RSH and RSH + RSSR
was measured at a wavelength of 412 nm using a SpectraMax
microplate reader (Molecular Devices, LLC, USA). A solution of cysteine was used for the preparation of a standard curve. The concentration of RSSR was calculated by the difference between the nal
concentration of thiol groups reduced by hydrochloric acid
(RSH + RSSR) and the initial concentration of the RSH sample.
2.11. Infection of R. prolixus by T. rangeli in the presence of reduced
glutathione
Insects were infected and dissected as described above, and
reduced glutathione was added to inactivated blood at a nal concentration of 1 mM before the blood meal. After 7 days of feeding,
the insects were dissected, the anterior and posterior midguts were
lysed, and the protozoan present in 10 ll aliquots of each compartment were counts using a Neubauer chamber. An average of 510
insects were utilized in each experiment in a total of 14 trials with
different cellular suspensions of T. rangeli.
2.12. Statistical analysis
All the experiments were performed in triplicate, with similar
results obtained from at least three separate cell suspensions.
The data were statistically analyzed using Students t test, with statistical signicance considered at p < 0.05.
3. Results
3.1. SOD activity is increased in insects infected with T. rangeli
As the anterior midgut and posterior midgut are the main gateways to the colonization of R. prolixus by T. rangeli, we evaluated
the activity of antioxidant enzymes in both compartments. The
enzyme SOD catalyzes the dismutation of superoxide to H2O2
and O2 (Scandalios, 2005).
Fig. 1 shows that there was a signicant increase in the overall
SOD activity present in the AM and PM tissue homogenates in the
days following insect feeding. However, this increase was even
higher in the insects that were infected with T. rangeli, and the
major difference between the groups was on the 5th day, at 38%
in AM (Fig. 1A) and 76% in PM (Fig. 1B). On the 7th day after infection, the SOD activity of both segments showed the same activity
as the control group.
3.2. Catalase and glutathione peroxidase activities are decreased in
insects infected with T. rangeli
As the SOD enzyme catalyzes the dismutation of superoxide
radical into H2O2, we also investigated both glutathione peroxidase
(GPx) and catalase, enzymes involved in the degradation of H2O2.
GPx is responsible for the reduction of H2O2 and hydroperoxides
using reduced glutathione as a hydrogen donor (Turrens, 2004),
whereas catalase reduces H2O2 into water and oxygen and is the
only enzyme that degrades hydrogen peroxide without the consumption of equivalents reducers (Scandalios, 2005).
Different from the SOD activity, we found that the catalase
activity was decreased in AM and PM in the days after feeding in
the control and infected insects. Furthermore, in the presence of
T. rangeli, the activity of the enzyme appeared to be inhibited in
these compartments in the early days after feeding. In the infected
group, the major enzyme inhibition occurred on the 3rd day after
feeding and was 70% reduced in both compartments in relation

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121

Fig. 1. SOD activity of R. prolixus infected with T. rangeli. SOD activity was measured in homogenates of the anterior midgut (A) and posterior midgut (B) of R. prolixus fed
articially in the absence (white bars) or presence of 5  106 cells/ml of T. rangeli (black bars), as described in Section 2. The values represent the mean standard error of at
least three independent experiments. p < 0.05 compared to the control.

to the control group. On the 5th day after feeding, the catalase
activity levels in both the AM and PM compartments were
increased in the insects infected with T. rangeli, reaching levels
comparable to those of the control insects (Fig. 2).
Similar to the catalase activity, the GPx activities present in AM
and PM decreased in the days after feeding in the control group,
and this decrease was more pronounced in AM compared to PM.
In the infected insects, the activity of GPx was approximately
50% inhibited on the 3rd day after feeding in both AM and PM
(Fig. 3).

3.3. Hydrogen peroxide production is increased in insects infected with

T. rangeli
Fig. 4 shows an increase in the H2O2 production in the infected
insects on the 3rd day after feeding, that can be explained by the
inhibition of catalase and GPx activities (Figs. 2 and 3). On the
3rd day after feeding the increased of H2O2 in the infected insects
was higher in PM compared to AM and on the 5th day after feeding
there was no difference between the groups, probably due the
increase of catalase activity in the infected group (Fig. 2).

Corroborating these results, the levels of thiol group oxidation

also appeared to be 50% higher in the infected group compared
to the control group. In this case, there was no notable difference
between the compartments (Fig. 6).

3.5. The effect of reduced glutathione on the proliferation of T. rangeli

Oxidative stress generated in the gut of insects infected with T.
rangeli can occur due to the immune response of the insect as a
protective mechanism against the proliferation of parasites within
these compartments because T. rangeli uses these tissues for entry
to the hemolymph. Accordingly, a more reduced environment with
lower levels of oxidative stress could favor the proliferation of T.
rangeli in these compartments. Thus, to conrm our hypothesis,
we evaluated the prole of T. rangeli proliferation in AM and PM
in the presence of reduced glutathione (GSH). After 7 days of feeding, the insects were dissected and anterior and posterior midguts
were lysed. Fig. 7 shows an increase in the number of parasites,
approximately 50%, in both segments when the insects were
infected in the presence of 1 mM GSH (Fig. 7).

4. Discussion
3.4. Increased lipid peroxidation and thiol group oxidation
Figs. 5 and 6 show that there was an increase in the levels of
lipid peroxidation and the oxidation of thiol groups in both compartments on the 1st, 2nd, and 3rd days after feeding, possibly as
a consequence of redox imbalance in the tissues. Additionally,
the lipid peroxidation levels were higher in PM than in AM. In
the infected group, the increase in lipid peroxidation was 50%
higher after the 3rd day of feeding in both compartments (Fig. 5).

During the infection of R. prolixus by T. rangeli, Whitten and colleagues (2001) found that short epimastigote forms of the parasite
stimulate increased superoxide radical production in the insect
hemolymph than long forms. Similarly, strains with increased
infective capacity, such as Choachi, induce a greater immune
response than less infective strains, such as H14 (Whitten et al.,
2001). However, little is known about the contribution of antioxidant enzymes to the detoxication of these radical agents during T.

Fig. 2. Catalase activity of R. prolixus infected with T. rangeli. Catalase activity was measured in homogenates of the anterior midgut (A) and posterior midgut (B) of R. prolixus
fed articially in the absence (white bars) or presence of 5  106 cells/ml of T. rangeli (black bars), as described in Section 2. The values represent the mean standard error of
at least three independent experiments. p < 0.05 compared to the control.

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Fig. 3. Glutathione peroxidase activity of R. prolixus infected with T. rangeli. GPx activity was measured in homogenates of the anterior midgut (A) and posterior midgut (B) of
R. prolixus fed articially in the absence (white bars) or presence of 5  106 cells/ml of T. rangeli (black bars), as described in Section 2. The values represent the
mean standard error of at least three independent experiments. p < 0.05 compared to the control.

Fig. 4. Hydrogen peroxide production by R. prolixus infected with T. rangeli. H2O2 production was measured in homogenates of the anterior midgut (A) and posterior midgut
(B) of R. prolixus fed articially in the absence (white bars) or presence of 5  106 cells/ml of T. rangeli (black bars), as described in Section 2. The values represent the
mean standard error of at least three independent experiments. p < 0.05 compared to the control.

Fig. 5. Lipid peroxidation in R. prolixus infected with T. rangeli. Lipid peroxidation was measured in homogenates of the anterior midgut (A) and posterior midgut (B) of R.
prolixus fed articially in the absence (white bars) or presence of 5  106 cells/ml of T. rangeli (black bars), as described in Section 2. The values represent the mean standard
error of at least three independent experiments. p < 0.05 compared to the control.

Fig. 6. Oxidation of thiol groups in the anterior and posterior midguts of R. prolixus infected with T. rangeli. The oxidation of thiol groups was measured in homogenates of the
anterior midgut (A) and posterior midgut (B) of R. prolixus fed articially in the absence (white bars) or presence of 5  106 cells/ml of T. rangeli (black bars), as described in
Section 2. The values represent the mean standard error of at least three independent experiments. p < 0.05 compared to the control.

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123

Fig. 7. Effect of reduced glutathione on the proliferation of T. rangeli in the anterior and posterior midgut of R. prolixus. R. prolixus nymphs were articially fed in the presence
of 5  106 cells/ml T. rangeli in the absence (white bars) or presence (black bars) of 1 mM GSH, as indicated on the abscissa. After 7 days of feeding, the insects were dissected,
and anterior and posterior midguts were lysed. (A) Anterior midgut, (B) posterior midgut. The values represent the mean standard error of at least seven independent
experiments. p < 0.05 compared to the control.

rangeli infection of R. prolixus and the mechanisms that these parasites utilize to overcome the host immune response.
Antioxidant enzymes, such as SOD, catalase, and GPx have been
described in various tissues of R. prolixus by Paes and Oliveira
(1999), Paes et al. (2001), and the activities of catalase and SOD
were found to be more prominent in the midgut of this insect.
Because the anterior midgut and posterior midgut are the main
gateways to the colonization of R. prolixus by T. rangeli, we measured the activity of these enzymes in both compartments.
The increase of SOD enzyme (Fig. 1) and the decrease of catalase
and GPx activities (Figs. 2 and 3, respectively) in the early days
after infection are in accordance with the increase of H2O2
observed in the 3rd day after infection shown in Fig. 4. GPx activity
is related to the metabolism of hydroperoxides and the protection
of cells against the formation of lipid peroxidation. In this sense,
due to an increase in the concentration of H2O2 as a consequence
of the increased activity of SOD and inhibition of GPx and catalase
activities, there may be an increase in lipid peroxidation and oxidation of thiol groups contained in the AM and PM cells of R. prolixus
infected with T. rangeli. Considering the above results, the largest
generation of oxidative stress should occur in the rst days after
feeding, as we observed an imbalance in the activities of antioxidant enzymes related to the metabolism of hydroperoxides during
this time. Indeed, the results in Figs. 5 and 6 indicate an increase in
the levels of lipid peroxidation and thiol group oxidation in both
compartments on the 1st, 2nd, and 3rd days after feeding.
The oxidative imbalance in the insect gut may be a protective
mechanism of the immune system to combat the presence of parasites, and several studies relate an increase in free radical production as a mechanism to limit the development of parasites in their
invertebrate hosts (Hao et al., 2003; Macleod et al., 2007; Souza
et al., 1997). An increase in H2O2 production was also observed
in A. gambiae infected with the malaria parasite; moreover, mosquitoes capable of producing more H2O2 in their gut showed
greater resistance to parasite colonization (Kumar et al., 2003). Thioredoxin reductase, an antioxidant enzyme that promotes the conversion of oxidized thioredoxin and can act together with the
glutathione system to regenerate reduced glutathione, is also
downregulated in T. infestans infected with T. cruzi (Buarque
et al., 2013).
The oxidation of thiol groups by H2O2 has the ability to modify
proteins containing cysteine, such as tyrosine phosphatases, which
have a cysteine in the catalytic site. This mechanism can activate
different types of signaling cascades and transcription factors
(Herrera-ortiz et al., 2010). Thus, the increased oxidation of thiol
groups may be related to the activation of signaling cascades, culminating in the activation of the immune system of the insect.
Because ROS production limits the infection and colonization of
R. prolixus by T. rangeli, it is expected that the addition of antioxi-

dant molecules, such as GSH, would contribute to an increased proliferation of these microorganisms in the gut of the insect vector. In
fact, Fig. 7 shows that GSH was effective in inducing the proliferation of T. rangeli in both segments of the insect gut.
In 2001 it was shown by microscope that the invasion of T. rangeli in the midgut cells occurs in the anterior portion of the posterior midgut (de Oliveira and de Souza, 2001). In the present work,
we show that in infected insects, the highest activity of SOD and
lowest activity of catalase with the highest production of H2O2
occurs in the PM compartment. We also show here that there
was a greater lipid peroxidation and oxidation of thiol groups in
the PM compartment of infected insects compared to the control
group. Thus, increased ROS production in the early days of T. rangeli infection in R. prolixus may constitute a primary line of protection until the immune system becomes fully active.
Acknowledgments
We thank Mr. Fabiano Ferreira Esteves, Mr. Edimilson Antnio
Pereira, and Ms. Rosangela Rosa de Arajo for their excellent technical assistance. This work was supported by grants from the Brazilian Agencies Conselho Nacional de Desenvolvimento Cientco e
Tecnolgico (CNPq), Coordenao de Aperfeioamento de Pessoal
de Nvel Superior (CAPES), Fundao de Amparo Pesquisa do
Estado do Rio de Janeiro (FAPERJ), and Institute of National Science
and Technology of Structural Biology and Bioimage (INCTBEB).
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