Experimental Parasitology 145 (2014) 118–124

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Experimental Parasitology

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Rhodnius prolixus: Modulation of antioxidant defenses by Trypanosomarangeli

http://dx.doi.org/10.1016/j.exppara.2014.08.0020014-4894/� 2014 Elsevier Inc. All rights reserved.

Abbreviations: SOD, superoxide dismutase; GPx, glutathione peroxidase; GSH,reduced glutathione.⇑ Corresponding authors at: Instituto de Bioquímica Médica, Universidade

Federal do Rio de Janeiro, Av. Carlos Chagas Filho, 373, Ilha do Fundão, Rio deJaneiro 21941-902, Brazil.

E-mail addresses: rocco@bioqmed.ufrj.br (N. Rocco-Machado), meyer@bioqmed.ufrj.br (J.R. Meyer-Fernandes).

Daniela Cosentino-Gomes, Nathália Rocco-Machado ⇑, José Roberto Meyer-Fernandes ⇑Institute of Medical Biochemistry, Federal University of Rio de Janeiro (UFRJ), CCS, Bloco H, Cidade Universitária, Ilha do Fundão, 21941-590 Rio de Janeiro, RJ, BrazilInstitute of National Science and Technology of Structural Biology and Bioimage (INCTBEB), CCS, Bloco H, Cidade Universitária, Ilha do Fundão, 21941-590 Rio de Janeiro, RJ, Brazil

h i g h l i g h t s

� Infection with T. rangeli increasesoxidative 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. prolixusmidgut.

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

a r t i c l e i n f o

Article history:Received 9 December 2013Received in revised form 7 May 2014Accepted 3 August 2014Available online 14 August 2014

Keywords:Trypanosoma rangeliRhodnius prolixusAntioxidant enzymesReactive oxygen speciesParasite–host interaction

a b s t r a c t

Trypanosoma rangeli is a protozoan parasite of insects and mammals that is challenged by the constantaction of reactive oxygen species, generated either by its own metabolism or through the host immuneresponse. The aim of this work was to investigate whether T. rangeli is able to modify the redox state of itsinsect vector, Rhodnius prolixus, through the modulation of such antioxidant enzymes as superoxide dis-mutase (SOD), catalase, and GPx present in the midgut of the insect. We verified that in R. prolixus fedwith blood infected with T. rangeli there is an increase in SOD activity in the anterior and posterior mid-guts. 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 fedblood. These changes in the redox state of the vector led to an increase in lipid peroxidation and thioloxidation levels in the anterior and posterior midgut tissues. We also verified that the addition of1 mM GSH in the blood meal of the infected insects increased the proliferation of these parasites by50%. These results suggest that there is an increase in oxidative stress in the insect gut during T. rangeliinfection, 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 isable to infect vertebrate and invertebrate hosts. However, in con-trast to Trypanosoma cruzi, the etiologic agent of Chagas disease,

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 northernSouth America, which poses some problems for diagnosis(Martínez et al., 1993), mainly because of a high immunologicalcross-reactivity between these two parasites (Basso et al., 2004;Labriola and Cazzulo, 1995). The life cycle of T. rangeli in its verte-brate host is poorly known. In contrast, after ingestion as a trypo-mastigote, T. rangeli is known to multiply as an epimastigote in themidgut of its invertebrate host, invade the hemolymph and hemo-cytes to continue growth, and complete development in the sali-vary gland, the site where metacyclogenesis occurs (Hoare, 1972).

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Protozoan parasites are susceptible to a large number of oxida-tive conditions, including the reactive oxygen species (ROS) thatare part of the immune system of the insect vector. ROS can alsobe produced during the breakdown of hemoglobin in the stomachof the insect vector as a consequence of the release of largeamounts of heme or as a byproduct of the parasite’s own aerobicmetabolism (Finzi et al., 2004).

Rhodnius prolixus is a hematophagus insect and a potentialtransmitter of many pathogens. Only insect species of the genusRhodnius present infective forms of T. rangeli in their salivaryglands, and these insects are highly susceptible to parasitemia(Garcia et al., 2009). The establishment of T. rangeli infection inthe gut and in the hemocoel of its vector insect is possibly regu-lated by a variety of chemical and physiological processes. As theintestine is the first environment for the establishment of T. rangeliinfection, the many enzymes, proteins, and products of blooddigestion present in this environment may have fundamental rolesin 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 anti-oxidant enzymes has been described as an indirect mechanism ofthe defense against pathogens (Dubovskiy et al., 2008; Wanget al., 2001). Aedes aegypti infected with the bacteria Wolbachia alsoshows an increase in the ROS levels. ROS up-regulation resulted inactivation of the Toll pathway, which mediated the antioxidantdefenses. This immune pathway is also responsible for activationof antimicrobial peptides (Pan et al., 2011). Lee et al. (2013) showsthat Drosophila melanogaster infected with pathogenic bacteriahave an increase in the ROS levels due to the activation of dual oxi-dase (DUOX), a member of nicotinamide adenine dinucleotidephosphate (NADPH) oxidase family. Symbiotic microbes havelower ROS-producing effects. Molina-Cruz et al. (2008) show thatgenetic differences in systemic hydrogen peroxide (H2O2) levelsbetween Anopheles gambiae strains have broad effects in theirimmune response to Plasmodium and other pathogens such as bac-teria. The higher the systemic ROS levels in a given strain, the bet-ter the mosquitoes survive a bacterial challenge. Dietarysupplementation with antioxidants (vitamin C or uric acid) candramatically reduce the mosquito’s ability to mount an effectiveantibacterial response. Anopheles albimanus infected with Plasmo-dium berghei produces nitric oxide and H2O2 in the midgut, thatfunction as signals for the activation the mosquito system immuneresponse (Herrera-Ortiz et al., 2010). Buarque et al. (2013) showsthat Triatoma infestans infected with T. cruzi exhibits a downregu-lation of the antioxidant enzyme thioredoxin reductase.

Our goal in this work is verify the changes on redox state of theinsect vector R. prolixus, including the modulation of antioxidantenzymes such as superoxide dismutase (SOD), catalase and gluta-thione peroxidase (GPx), as well as, the level of H2O2 in the midgutof 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 thepreparation of all solutions was filtered using a four-stage Milli-Qsystem (Millipore Corp., Bedford, MA, USA).

2.2. Microorganisms

Epimastigote forms of T. rangeli strain H14 (supplied by Dr.Maria Auxiliadora Sousa, from Coleção de Tripanossomatídeos,

Instituto Oswaldo Cruz, Rio de Janeiro, Brazil) were maintainedat 28 �C in liver infusion tryptose (LIT) medium adjusted to pH7.2 with HCl and supplemented with 20% fetal bovine serum. Theparasites were grown for 7 days in culture medium, collected bycentrifugation at 1500�g at 4 �C for 15 min, and washed threetimes in a buffer solution containing 50 mM Tris–HCl (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 main-tained at 28 �C and relative humidity of 70–80% under a photope-riod of 12 h light/12 h dark. Adult insects were fed live rabbit bloodat 3-week intervals, beginning 15 days after molting. Animal careand experimental protocols were approved by the Committee forEvaluation of Animal Use for Research of Federal University ofRio de Janeiro (CAUAP-UFRJ), under the registry #IBQM001.

Male insects at the 5th stage and fasted for at least 4 weekswere randomly selected and fed human blood containing citratevia artificial feeding. For infection, 5 � 106 cells/ml of T. rangeliepimastigotes were collected in the stationary phase and addedto human blood previously inactivated for 1 h at 56 �C, as previ-ously described by Gonzalez et al., 2013. After an incubation per-iod, the insects were dissected to remove portions of the anteriorand posterior midguts (Gomes et al., 2003).

2.4. Dissection of R. prolixus

After the insects were dissected, the anterior and posterior mid-guts were isolated and washed in phosphate-buffered saline (PBS),pH 7.4, to remove all of the contents of the lumen, as described byGrillo et al. (2007). The two intestinal segments were then homog-enized in the same buffer containing a cocktail of protease inhibi-tors (Sigma) and 1 mM of phenylmethylsulfonyl chloride (PMSF).We used 200 ll and 150 ll of the buffer to homogenize the ante-rior and posterior midguts, respectively. The samples were centri-fuged at 11,000�g for 5 min, and the supernatants were used forthe enzyme activity measurements. The total protein concentra-tion was determined by the method of Lowry et al. (1951) usingbovine serum albumin as the standard.

2.5. Catalase assay

To measure catalase activity, 0.05 mg/ml of protein was addedto a reaction medium containing 100 mM Tris–HCl (pH 7.4). Thereaction was initiated by the addition of the substrate, 8 mMH2O2. The dismutation of H2O2 was monitored in a quartz cuvetteusing a SpectraMax microplate reader (Molecular Devices, LLC,USA) at a wavelength of 240 nm for 300 s, as described by Paeset al. (2001). The catalase activity was calculated using the molarextinction coefficient of H2O2, 36 M�1 � cm�1 and is expressed inmmol of H2O2 ptn �mg�1 �min�1.

2.6. Glutathione peroxidase assay

To measure GPx activity, 0.05 mg/ml of protein was added to areaction medium containing PBS (pH 7.4), 10 mM sodium azide,1 mM EDTA, 1 mM GSH, 0.1 mM NADPH, 2 U/ml glutathionereductase; 0.25 mM H2O2 was used as the substrate. The sample(10 lL) was pre-incubated in the medium, and the reaction wasinitiated by the addition of hydrogen peroxide. The oxidation ofNADPH was monitored in a quartz cuvette using a SpectraMaxmicroplate reader (Molecular Devices, LLC, USA) at a wavelengthof 340 nm for 300 s; readings were obtained every 30 s, asdescribed by Paes and Oliveira (1999). The GPx activity was calcu-lated by subtracting the absorbance of the blank (in the absence of

120 D. Cosentino-Gomes et al. / Experimental Parasitology 145 (2014) 118–124

sample) and using the molar extinction coefficient of NADPH,6.22 mM�1 � cm�1; the activity is expressed in nmol of NADPHptn�1 �mg�1 �min.

2.7. Superoxide dismutase assay

For measurement of the SOD activity, a 20-lL aliquot of thesample 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 reac-tion was initiated by the addition of 20 lL of xanthine to the reac-tion medium in a 96-well plate. Readings were obtained followingthe reduction of cytochrome c at 550 nm using a SpectraMaxmicroplate reader (Molecular Devices, LLC, USA), with 300 readingsevery 30 s. This method uses the xanthine-xanthine oxidase sys-tem as a generator of superoxide radicals, which are used for theoxidation of cytochrome c. The activity of superoxide dismutaseis given by the ability of this enzyme to compete with cytochromec for superoxide. Thus, one unit of SOD activity corresponds to a50% inhibition of the reaction between superoxide and cytochromec (Mccord and Fridovich, 1969; Kabil et al., 2007).

2.8. Hydrogen peroxide production

To measure the H2O2 production, we used homogenates of theanterior (AM) and posterior midgut (PM) of 5 insects under eachcondition from 3rd and 5th days. The homogenates were kept onice throughout the procedure. After maceration, supernatant sam-ples 0.05 mg protein/ml were incubated for 20 min in a mediumcontaining PBS (pH 7.4), 1.7 lM Amplex Red and 6.7 U/ml horse-radish peroxidase (HPR) in a final volume of 200 ll (Meyer et al.,2006). HRP catalyzes the reaction between Amplex Red and H2O2

forming the fluorophore resorufin, which was monitored at excita-tion and emission wavelengths of 563 (slit 5 nm) and 587 nm (slit5 nm), respectively. After 20 min of reaction, H2O2 production wasthen determined using a standard curve with known quantities ofH2O2.

2.9. Lipid peroxidation

The thiobarbituric acid method (TBARS), as described by Vynce(1970), was utilized to measure lipid peroxidation. We usedhomogenates of the anterior (AM) and posterior midgut (PM) of8 insects under each condition. The homogenates were kept onice throughout the procedure. After maceration, the tissue sampleswere incubated for 15 min in 10% trichloroacetic acid at a ratio of2:1 acid:sample. The tubes were centrifuged at 2200�g for 15 minat 4 �C, and 400 ll of the supernatant was removed and added toan equal volume of 0.67% thiobarbituric acid (TBA). The sampleswere incubated for 15 min at 100 �C, and 200 ll of each samplewas placed in a well of 96-well plates and measured at 532 nmusing a SpectraMax microplate reader (Molecular Devices, LLC,USA). The levels of lipid peroxidation were calculated from thequantification of the malondialdehyde (MDA) formed by meansof a standard curve using known concentrations of 1,1,3,3-tetra-methoxypropane (Sigma) and normalized to the protein concen-tration of each sample.

2.10. Quantification of oxidized thiol groups

The method based on the oxidation of RSH by 5,50-dito-bis(2-nitrobenzoic 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 ofAM and PM homogenates were incubated with 1 mM hydrochloricacid 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 ofthe decomposed sample were incubated with 500 ll of 0.1% DTNBin PBS for 10 min at 37 �C. The absorbance of RSH and RSH + RSSRwas measured at a wavelength of 412 nm using a SpectraMaxmicroplate reader (Molecular Devices, LLC, USA). A solution of cys-teine was used for the preparation of a standard curve. The concen-tration of RSSR was calculated by the difference between the finalconcentration 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 reducedglutathione

Insects were infected and dissected as described above, andreduced glutathione was added to inactivated blood at a final con-centration of 1 mM before the blood meal. After 7 days of feeding,the insects were dissected, the anterior and posterior midguts werelysed, and the protozoan present in 10 ll aliquots of each compart-ment were counts using a Neubauer chamber. An average of 5–10insects were utilized in each experiment in a total of 14 trials withdifferent cellular suspensions of T. rangeli.

2.12. Statistical analysis

All the experiments were performed in triplicate, with similarresults obtained from at least three separate cell suspensions.The data were statistically analyzed using Student’s t test, with sta-tistical significance 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 gate-ways to the colonization of R. prolixus by T. rangeli, we evaluatedthe activity of antioxidant enzymes in both compartments. Theenzyme SOD catalyzes the dismutation of superoxide to H2O2

and O2 (Scandalios, 2005).Fig. 1 shows that there was a significant increase in the overall

SOD activity present in the AM and PM tissue homogenates in thedays following insect feeding. However, this increase was evenhigher in the insects that were infected with T. rangeli, and themajor 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 infec-tion, the SOD activity of both segments showed the same activityas the control group.

3.2. Catalase and glutathione peroxidase activities are decreased ininsects infected with T. rangeli

As the SOD enzyme catalyzes the dismutation of superoxideradical 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 hydroperoxidesusing reduced glutathione as a hydrogen donor (Turrens, 2004),whereas catalase reduces H2O2 into water and oxygen and is theonly enzyme that degrades hydrogen peroxide without the con-sumption of equivalents reducers (Scandalios, 2005).

Different from the SOD activity, we found that the catalaseactivity was decreased in AM and PM in the days after feeding inthe control and infected insects. Furthermore, in the presence ofT. rangeli, the activity of the enzyme appeared to be inhibited inthese compartments in the early days after feeding. In the infectedgroup, the major enzyme inhibition occurred on the 3rd day afterfeeding and was 70% reduced in both compartments in relation

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 fedartificially 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 atleast three independent experiments. ⁄p < 0.05 compared to the control.

D. Cosentino-Gomes et al. / Experimental Parasitology 145 (2014) 118–124 121

to the control group. On the 5th day after feeding, the catalaseactivity levels in both the AM and PM compartments wereincreased in the insects infected with T. rangeli, reaching levelscomparable to those of the control insects (Fig. 2).

Similar to the catalase activity, the GPx activities present in AMand 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 approximately50% inhibited on the 3rd day after feeding in both AM and PM(Fig. 3).

3.3. Hydrogen peroxide production is increased in insects infected withT. rangeli

Fig. 4 shows an increase in the H2O2 production in the infectedinsects on the 3rd day after feeding, that can be explained by theinhibition of catalase and GPx activities (Figs. 2 and 3). On the3rd day after feeding the increased of H2O2 in the infected insectswas higher in PM compared to AM and on the 5th day after feedingthere was no difference between the groups, probably due theincrease of catalase activity in the infected group (Fig. 2).

3.4. Increased lipid peroxidation and thiol group oxidation

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

Fig. 2. Catalase activity of R. prolixus infected with T. rangeli. Catalase activity was measufed artificially in the absence (white bars) or presence of 5 � 106 cells/ml of T. rangeli (blaat least three independent experiments. ⁄p < 0.05 compared to the control.

Corroborating these results, the levels of thiol group oxidationalso appeared to be 50% higher in the infected group comparedto the control group. In this case, there was no notable differencebetween 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 aprotective mechanism against the proliferation of parasites withinthese compartments because T. rangeli uses these tissues for entryto the hemolymph. Accordingly, a more reduced environment withlower levels of oxidative stress could favor the proliferation of T.rangeli in these compartments. Thus, to confirm our hypothesis,we evaluated the profile of T. rangeli proliferation in AM and PMin the presence of reduced glutathione (GSH). After 7 days of feed-ing, the insects were dissected and anterior and posterior midgutswere lysed. Fig. 7 shows an increase in the number of parasites,approximately 50%, in both segments when the insects wereinfected in the presence of 1 mM GSH (Fig. 7).

4. Discussion

During the infection of R. prolixus by T. rangeli, Whitten and col-leagues (2001) found that short epimastigote forms of the parasitestimulate increased superoxide radical production in the insecthemolymph than long forms. Similarly, strains with increasedinfective capacity, such as Choachi, induce a greater immuneresponse than less infective strains, such as H14 (Whitten et al.,2001). However, little is known about the contribution of antioxi-dant enzymes to the detoxification of these radical agents during T.

red in homogenates of the anterior midgut (A) and posterior midgut (B) of R. prolixusck bars), as described in Section 2. The values represent the mean ± standard error of

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) ofR. prolixus fed artificially 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 themean ± 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 artificially 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 themean ± 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 artificially 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 ± standarderror 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 theanterior midgut (A) and posterior midgut (B) of R. prolixus fed artificially in the absence (white bars) or presence of 5 � 106 cells/ml of T. rangeli (black bars), as described inSection 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. 7. Effect of reduced glutathione on the proliferation of T. rangeli in the anterior and posterior midgut of R. prolixus. R. prolixus nymphs were artificially fed in the presenceof 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 independentexperiments. ⁄p < 0.05 compared to the control.

D. Cosentino-Gomes et al. / Experimental Parasitology 145 (2014) 118–124 123

rangeli infection of R. prolixus and the mechanisms that these par-asites utilize to overcome the host immune response.

Antioxidant enzymes, such as SOD, catalase, and GPx have beendescribed in various tissues of R. prolixus by Paes and Oliveira(1999), Paes et al. (2001), and the activities of catalase and SODwere found to be more prominent in the midgut of this insect.Because the anterior midgut and posterior midgut are the maingateways to the colonization of R. prolixus by T. rangeli, we mea-sured the activity of these enzymes in both compartments.

The increase of SOD enzyme (Fig. 1) and the decrease of catalaseand GPx activities (Figs. 2 and 3, respectively) in the early daysafter infection are in accordance with the increase of H2O2

observed in the 3rd day after infection shown in Fig. 4. GPx activityis related to the metabolism of hydroperoxides and the protectionof cells against the formation of lipid peroxidation. In this sense,due to an increase in the concentration of H2O2 as a consequenceof the increased activity of SOD and inhibition of GPx and catalaseactivities, there may be an increase in lipid peroxidation and oxida-tion of thiol groups contained in the AM and PM cells of R. prolixusinfected with T. rangeli. Considering the above results, the largestgeneration of oxidative stress should occur in the first days afterfeeding, as we observed an imbalance in the activities of antioxi-dant enzymes related to the metabolism of hydroperoxides duringthis time. Indeed, the results in Figs. 5 and 6 indicate an increase inthe levels of lipid peroxidation and thiol group oxidation in bothcompartments on the 1st, 2nd, and 3rd days after feeding.

The oxidative imbalance in the insect gut may be a protectivemechanism of the immune system to combat the presence of par-asites, and several studies relate an increase in free radical produc-tion as a mechanism to limit the development of parasites in theirinvertebrate hosts (Hao et al., 2003; Macleod et al., 2007; Souzaet al., 1997). An increase in H2O2 production was also observedin A. gambiae infected with the malaria parasite; moreover, mos-quitoes capable of producing more H2O2 in their gut showedgreater resistance to parasite colonization (Kumar et al., 2003). Thi-oredoxin reductase, an antioxidant enzyme that promotes the con-version of oxidized thioredoxin and can act together with theglutathione system to regenerate reduced glutathione, is alsodownregulated in T. infestans infected with T. cruzi (Buarqueet al., 2013).

The oxidation of thiol groups by H2O2 has the ability to modifyproteins containing cysteine, such as tyrosine phosphatases, whichhave a cysteine in the catalytic site. This mechanism can activatedifferent types of signaling cascades and transcription factors(Herrera-ortiz et al., 2010). Thus, the increased oxidation of thiolgroups may be related to the activation of signaling cascades, cul-minating in the activation of the immune system of the insect.

Because ROS production limits the infection and colonization ofR. prolixus by T. rangeli, it is expected that the addition of antioxi-

dant molecules, such as GSH, would contribute to an increased pro-liferation of these microorganisms in the gut of the insect vector. Infact, Fig. 7 shows that GSH was effective in inducing the prolifera-tion of T. rangeli in both segments of the insect gut.

In 2001 it was shown by microscope that the invasion of T. ran-geli in the midgut cells occurs in the anterior portion of the poster-ior midgut (de Oliveira and de Souza, 2001). In the present work,we show that in infected insects, the highest activity of SOD andlowest activity of catalase with the highest production of H2O2

occurs in the PM compartment. We also show here that therewas a greater lipid peroxidation and oxidation of thiol groups inthe PM compartment of infected insects compared to the controlgroup. Thus, increased ROS production in the early days of T. ran-geli infection in R. prolixus may constitute a primary line of protec-tion until the immune system becomes fully active.

Acknowledgments

We thank Mr. Fabiano Ferreira Esteves, Mr. Edimilson AntônioPereira, and Ms. Rosangela Rosa de Araújo for their excellent tech-nical assistance. This work was supported by grants from the Bra-zilian Agencies Conselho Nacional de Desenvolvimento Científico eTecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoalde Nível Superior (CAPES), Fundação de Amparo à Pesquisa doEstado do Rio de Janeiro (FAPERJ), and Institute of National Scienceand Technology of Structural Biology and Bioimage (INCTBEB).

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