Antibacterial activity using inhibition zone (ZI) assay in <i>Rhodnius prolixus</i> 5^th-instar nymphs infected with <i>Trypanosoma cruzi</i> Dm28c clone.

<p>(A) Antibacterial activity with anterior and posterior midgut regions at 9 days after feeding; (B) Antibacterial activity of anterior midgut at 5, 9 and 16 days after feeding. Bars represent mean ± SEM. Means were analyzed by using t Test and 1 way ANOVA. Each bar represents the mean (+/−SEM) of four separate experiment, n = 6−10 insects for each determination.</p

Phenoloxidase activity (A) and nitrite and nitrate production (B) in <i>Rhodnius prolixus</i> 5^th-instar nymphs challenged by <i>Trypanosoma cruzi</i> Dm28c clone. Anterior midgut samples collected nine days after feeding and infection.

<p>Treatments: C – control insects fed on blood alone; CC – parasite infected insects; A – insects treated antibiotic; AC- insects treated with antibiotic and infected with the parasite. Bars represent mean ± SEM. Means were analyzed by using 1 way ANOVA comparing all groups to the control (C) and all groups to the insects treated with antibiotic (A). Each experiment represents the mean (+/−SEM) of four separate experiment, n = 6–10 insects for each determination; p<0.001, *** extremely significant, ** very significant and * significant.</p

Parasite infection and microbiota population in <i>Rhodnius prolixus</i> 5^th-instar nymphs digestive tract challenged by <i>Trypanosoma cruzi</i> Dm28c clone.

<p>(A) Parasite infection at different days after infection. (B) Microbiota population at different days after feeding. (C) Microbiota population 8 days after feeding. Treatments: C – control insects fed on blood alone; CC – insects infected with <i>T. cruzi</i> Dm28c clone; A – insects treated with antibiotic alone; AC- insects treated with antibiotic and infected with the Dm28c parasites; Y- insects infected with <i>T. cruzi</i> Y strain; AY–insects treated with antibiotic and infected with <i>T. cruzi</i> Y strain. In figures A and C each point represents the number of parasites or bacteria in an individual digestive tract, and horizontal lines indicate the median. In figure B each point represents the median. In figure C the median for antibiotic treated and infected insects (AC) is zero and therefore overlaps the x axes. Treatments were repeated 3–5 times with 6–10 insects in each experiment reaching a total of 25 to 35 insects for each group. Medians were analyzed with 1 way ANOVA and Mann Whitney test.</p

Antibacterial activity in <i>Rhodnius prolixus</i> 5^th-instar nymphs challenged by <i>Trypanosoma cruzi</i> Dm28c clone.

<p><b>Anterior midgut samples collected nine days after feeding and infection.</b> (A) Inhibition zone (ZI) assay incubated for 24 h at 37°C. (B) Turbidometric (TB) assay incubated for 11 h with readings at each hour. (C) Turbidometric (TB) assay after 4 h of incubation of uninfected or infected control insects. (D) Turbidometric assay after 11 h of incubation of insects treated with antibiotic alone or with antibiotic and then infected. Treatments: C – control insects fed on blood alone; CC – parasite infected insects; A – insects treated with antibiotic; AC- insects treated with antibiotic and then infected with parasites. Each bar represents mean ± SEM of four experiments, n = 6–10 insects for each determination. Means were analyzed by using t Test, Mann Whitney test and 2 way ANOVA.</p

Effect of surface mucins on <i>ex vivo T. cruzi</i> attachment to the midgut epithelium of <i>Rhodnius prolixus</i>.

<p>Midguts obtained from male fifth-instar nymphs 10 days after the bloodmeal were previously incubated for 30 min in PBS supplemented with the indicated mucin peptides and added with BHI interaction medium containing flagellates (2.5×10⁷/ml). Pre-incubation with mucin peptides was omitted in control (non-treated) group. Adhered epimastigotes were counted per 100 epithelial cells in 10 different fields of each midgut preparation. (A) Pre-incubation in 1 µg/ml of TcSMUG S, TSSA or TcSMUG L. (B) Pre-incubation in 0.01, 0.1 or 1.0 µg/ml of TcSMUG L. (C) Pre-incubation in 0.01, 0.1 or 1.0 µg/ml of TSSA. Each group represents mean ± S.D. of parasites attached in 10 midguts. Asterisk represents experimental groups with statistical significance compared to the control. <i>Trypanosoma cruzi</i> small mucin S (TcSMUG S), <i>Trypanosoma cruzi</i> small mucin L (TcSMUG L) and trypomastigote small surface antigen (TSSA).</p

Effect of surface mucins on <i>T. cruzi in vivo</i> development in the digestive tract of <i>Rhodnius prolixus</i>.

<p>Insects were fed on citrated, complement-inactivated human blood containing 2×10⁵ flagellates/ml. Each mucin peptide was added to the bloodmeal at a concentration of 30 µg/ml and insects dissected as days 7, 14 or 21 post feeding. Each point represents mean±S.D of flagellates/ml in the whole gut of 10 insects. Asterisk represents experimental groups with statistical significance compared to the control.</p

Western blots of <i>TcSMUG L</i> products from <i>T. cruzi</i>.

<p>A) Extracts of epimastigotes from different parasite stocks (Ad, Adriana; CL, CL Brener; Dm, Dm28c) were probed with either anti-TcSMUG L antibodies or anti-glutamate dehydrogenase (GDH) antiserum. B) ConA-fractionated extracts of Dm28c epimastigotes were probed with anti-TcSMUG L antiserum. ft, flow-through. C) Butan-1-ol extraction analysis of Dm28c delipidated epimastigotes. Fractions, named according to <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002552#pntd.0002552-Almeida1" target="_blank">[19]</a>, were probed with affinity-purified anti-TcSMUG L antibodies. Molecular mass markers (in kDa) are indicated at right. *Denotes aggregates.</p

Photomicrographs of posterior midgut epithelial cells of fifth-instar <i>R. prolixus</i> incubated with biotin-labeled peptides.

<p>(A) Light microscopy showing single-globe columnar epithelial cells(white star) and PMM (white arrow). (B) Fluorescence microscopy showing that no demarcation was observed after incubation with avidin-FITC-labeled conjugate alone. Light and fluorescence microscopy, respectively, of samples incubated with biotin-labeled TcSMUG L (C and D), biotin-labeled TcSMUG S (E and F), and biotin-labeled TSSA (G and H). Fluorescence of the surface and nucleolus of the midgut cells is indicated by white and black arrows (respectively). 400×.</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

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