Real-time PCR analysis of parasite loads during R. prolixus infection: the effect of antioxidants in vivo 5 dpi.

<p>Fifth instar <i>R</i>. <i>prolixus</i> nymphs were fed serum-inactivated rabbit blood or blood supplemented with 1 mM NAC or 1 mM urate and 5 x 10⁷ epimastigotes/mL (at least ten insects per group in each experiment). Five days post infection, the bugs were dissected, and the total RNA of the <b>(A)</b> anterior midgut, <b>(B)</b> posterior midgut or <b>(C)</b> the rectum was extracted in TRIZOL reagent. A cDNA strand was synthetized and used as a template for amplification with TCZ primers. RpMIP was used as an endogenous control, ^*p<0.05 compared with the blood group by one-way ANOVA and Tukey’s test.</p

The effect of molecules of distinct redox status upon metacyclogenesis <i>in vitro</i>.

<p><i>T</i>. <i>cruzi</i> epimastigotes were incubated in TAU3AAG medium containing 30 μM GSH, 30 μM NAC,1 mM urate, 30 μM heme or 30 μM β-hematin, as described in the Materials and Methods. <b>(A)</b> Culture supernatants were collected at different time periods, and the percentage of total parasites in the supernatant after 96 h treatment was calculated. The parasite evolutive forms were determined by light microscopy according to the kinetoplast position. <b>(B)</b> The data represent means ± standard errors of the percentage of trypomastigotes from five independent experiments. <b>(C)</b> The data represent means ± standard errors of the percentage of epimastigotes from three independent experiments, ^*p<0.05 compared with the control group by one-way ANOVA and Tukey’s test.</p

Effects of molecules of distinct redox status on epimastigote proliferation <i>in vitro</i>.

<p><i>T</i>. <i>cruzi</i> epimastigotes (2.5 x 10⁶cells/mL) were incubated in BHI medium supplemented with 10% FCS in the absence (control) or in the presence of 30 μM heme, <b>(A)</b> 30 μM β-hematin; <b>(B)</b> different concentrations of GSH (30 μM or 1 mM) in the absence or presence of 30 μM heme; or with <b>(C)</b> different concentrations of NAC (30 μM or 1 mM) in the absence or presence of 30 μM heme. All data are presented as the means ± standard deviation. Statistical analysis was performed for the 12^th day of treatment, ^*p<0.05 compared with the control group and ^#p<0.05 compared with heme treatment by one-way ANOVA and Tukey’s test.</p

Heme crystallization in a Chagas disease vector acts as a redox-protective mechanism to allow insect reproduction and parasite infection

<div><p>Heme crystallization as hemozoin represents the dominant mechanism of heme disposal in blood feeding triatomine insect vectors of the Chagas disease. The absence of drugs or vaccine for the Chagas disease causative agent, the parasite <i>Trypanosoma cruzi</i>, makes the control of vector population the best available strategy to limit disease spread. Although heme and redox homeostasis regulation is critical for both triatomine insects and <i>T</i>. <i>cruzi</i>, the physiological relevance of hemozoin for these organisms remains unknown. Here, we demonstrate that selective blockage of heme crystallization <i>in vivo</i> by the antimalarial drug quinidine, caused systemic heme overload and redox imbalance in distinct insect tissues, assessed by spectrophotometry and fluorescence microscopy. Quinidine treatment activated compensatory defensive heme-scavenging mechanisms to cope with excessive heme, as revealed by biochemical hemolymph analyses, and fat body gene expression. Importantly, egg production, oviposition, and total <i>T</i>. <i>cruzi</i> parasite counts in <i>R</i>. <i>prolixus</i> were significantly reduced by quinidine treatment. These effects were reverted by oral supplementation with the major insect antioxidant urate. Altogether, these data underscore the importance of heme crystallization as the main redox regulator for triatomine vectors, indicating the dual role of hemozoin as a protective mechanism to allow insect fertility, and <i>T</i>. <i>cruzi</i> life-cycle. Thus, targeting heme crystallization in insect vectors represents an innovative way for Chagas disease control, by reducing simultaneously triatomine reproduction and <i>T</i>. <i>cruzi</i> transmission.</p></div

Oogenesis and <i>Trypanosoma cruzi</i> infection depend on heme crystallization in the midgut.

<p>Insects were fed with blood (Ctrl) or blood supplemented with 100 μM QND or blood supplemented with 100 μM QND and 1 mM urate (QND+urate). <b>(A)</b> Stereoscope images of adult female abdomens from IBqM colony four days after feeding. Ovaries are depicted as dashed white lines. <b>(B)</b> The average number of eggs laid per females from Fiocruz colony insects fed with blood was determined along 24 days after blood meal. Ctrl: n≥3; 100 μM QND: n≥3; QND+urate: n≥3. Comparisons between groups were done by two-way ANOVA and a <i>posteriori</i> Bonferroni’s tests (^ap<0.0001 relative to QND and QND+urate, and ^bp<0.0001 relative to QND). <b>(C)</b> Total <i>T</i>. <i>cruzi</i> counts in the digestive tract of infected adult females from Fiocruz colony 15 days after blood meal. Ctrl: n = 18; 100 μM QND: n = 18; QND + urate: n = 8). Comparisons between groups were done by one-way ANOVA, (*<i>p</i> = 0.007 relative to Ctrl), with a <i>posteriori</i> Bonferroni’s tests. Data in Fig 4B were expressed as mean ± S.E.M., and in Fig 4C as scattered plot with gray lines representing medians.</p

Limited Hz formation causes systemic heme overload, activation of compensatory heme detoxification mechanisms, and redox imbalance.

<p>Insects were fed with blood (Control, Ctrl), or blood supplemented with quinidine (QND) and analyzed four days (<b>A-E,G</b>) or along fifteen days (<b>F</b>) after feeding. All experiments were conducted using insects from IBqM colony. <b>(A)</b> Light absorption spectra of hemolymph. Insets show the dark reddish-color of hemolymph from control and QND treated insects. <b>(B)</b> Total heme concentrations in the hemolymph. Ctrl: n = 17; 100 μM QND: n = 16. Comparisons between groups were done by Student’s t test (*p<0.01). <b>(C)</b> Relative expression of <i>Rhodnius</i> heme-binding protein (RHBP) in fat bodies. Ctrl: n = 3; 100 μM QND: n = 3. Comparisons between groups were done by Student’s t test (*p<0.005 relative to Ctrl). <b>(D)</b> Heme buffering capacity of hemolymph from the insects. Ctrl: n≥4; 100 μM QND: n≥3. Comparisons between groups were done by two-way ANOVA and <i>a posteriori</i> Bonferroni’s tests (*p<0.05). <b>(E)</b> Lipid peroxide levels in the hemolymph. Ctrl: n = 4; QND: n≥6. Comparisons between groups were done by one-way ANOVA and <i>a posteriori</i> Tukey’s tests (*p<0.05 relative to Ctrl). <b>(F)</b> Urate levels in the hemolymph. Ctrl: n≥3; 100 μM QND: n≥3. Comparisons between groups were done by two-way ANOVA and <i>a posteriori</i> Bonferroni’s tests (*p<0.05 relative to Ctrl). <b>(G)</b> Urate levels in the hemolymph from insects fed with saline (Ctrl, n = 8), saline supplemented with 100 μM QND (n = 9), blood (Ctrl, n = 44), or blood supplemented with 100 μM QND (n = 46). Comparisons between groups were done by Mann Whitney´s test (*p<0.0005 relative to Blood Ctrl). Data in Figs 3B-3G were expressed as mean ± S.E.M.</p

Impaired heme crystallization affects posterior midgut organization.

<p><b>(A and B)</b> Light microscopy images of posterior midguts of adult insects fed with blood (Ctrl; <b>A</b>) and blood supplemented with 100 μM QND (<b>B</b>) from IBqM colony four days after blood meal (bars = 20 μm). Midgut lumen (ML), midgut cells (Mc), hemocoel (Hc), vacuoles (black arrows) and lipid droplets (white arrows) are indicated in the images. <b>(C-H)</b> Transmission electron microscopy images of posterior midguts from insects maintained at IBqM (<b>C-F</b>), and Fiocruz (<b>G,H</b>) colonies fed with blood (Ctrl, <b>C,F</b>), or blood + 100 μM QND (<b>D,E,G,H</b>) four days after blood meal. The general architecture of posterior midgut cells in control insects (<b>C,F</b>) includes mitochondria (white arrows), endoplasmic reticulum (dashed box) and microvilli (asterisks). In QND treated insects (<b>D,E,G,H</b>), extensive organelle disappearance contrasts with the presence of numerous electron-dense hemoxisomes/residual bodies (arrowheads), vacuoles (black arrow), and intracellular lipid droplets (LD). Electron-dense mitochondria found in the posterior midgut of control insects (<b>F</b>), contrast with swollen and washed out mitochondria from 100 μM QND treated insects (<b>G</b>). <b>(H)</b> Mitochondria inside an autophagosome. (Scale bars: C-E: 2 μm; F: 1.0 μm; G,H: 0.5 μm).</p

Schematic model of physiological consequences of blocked Hz formation in triatomine midgut.

<p>In the presence of QND, heme derived from blood meal forms stable complexes with this drug, impairing Hz formation in the midgut lumen. Non-crystallized heme levels build up in the midgut causing cytotoxic effects to <i>T</i>. <i>cruzi</i> trypomastigotes. Excessive heme is transported to hemolymph through the midgut cells by hemoxisomes/residual bodies, causing redox imbalance and autophagy in the midgut. Heme accumulates in the hemolymph, increasing RHBP production, as a compensatory defense against "free" heme. However, this mechanism is overwhelmed, as the levels of urate drop. Redox imbalance has a direct effect on oogenesis, reducing egg production.</p

Quinidine inhibits heme crystallization <i>in vivo</i> and causes redox imbalance in triatomine posterior midgut.

<p>Insects were fed with blood (Control, Ctrl) or blood supplemented with quinidine (QND). <b>(A)</b> Posterior midgut Hz content four days after feeding. Ctrl: n = 8; QND: n≥3. Comparisons between groups were done by one-way ANOVA and <i>a posteriori</i> Tukey’s tests (*p<0.05 relative to Ctrl). <b>(B)</b> Hz content in posterior midgut along 15 days after blood meal. Ctrl: n = 8; 100 μM QND: n≥3. Comparisons between groups were done by two-way ANOVA and <i>a posteriori</i> Bonferroni’s tests (<i>B</i>; *p<0.05 relative to Ctrl). <b>(C)</b> Intracellular oxidants levels assessed by fluorescence microscopy of dihydroethidium (DHE) stained posterior midguts four days after feeding (400x magnification). Insets are bright field images of the same midgut regions. <b>(D)</b> The average DHE fluorescence intensity in the posterior midgut of insects. Ctrl: n = 24; 100 μM QND: n = 35). Comparisons between groups were done by Mann Whitney’s test (D; *p<0.0005 relative to Ctrl). All data are expressed as mean ± S.E.M. and experiments were conducted using adult insects only from IBqM colony.</p