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

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

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 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

Metabolic Signatures of Triatomine Vectors of <i>Trypanosoma cruzi</i> Unveiled by Metabolomics

<div><p>Chagas disease is a trypanosomiasis whose causative agent is the protozoan parasite <i>Trypanosoma cruzi</i>, which is transmitted to humans by hematophagous insects known as triatomines and affects a large proportion of South America. The digestive tract of the insect vectors in which <i>T. cruzi</i> develops constitutes a dynamic environment that affects the development of the parasite. Thus, we set out to investigate the chemical composition of the triatomine intestinal tract through a metabolomics approach. We performed Direct Infusion Fourier Transform Ion Cyclotron Resonance Mass Spectrometry on fecal samples of three triatomine species (<i>Rhodnius prolixus</i>, <i>Triatoma infestans</i>, <i>Panstrongylus megistus</i>) fed with rabbit blood. We then identified groups of metabolites whose frequencies were either uniform in all species or enriched in each of them. By querying the Human Metabolome Database, we obtained putative identities of the metabolites of interest. We found that a core group of metabolites with uniform frequencies in all species represented approximately 80% of the molecules detected, whereas the other 20% varied among triatomine species. The uniform core was composed of metabolites of various categories, including fatty acids, steroids, glycerolipids, nucleotides, sugars, and others. Nevertheless, the metabolic fingerprint of triatomine feces differs depending on the species considered. The variable core was mainly composed of prenol lipids, amino acids, glycerolipids, steroids, phenols, fatty acids and derivatives, benzoic acid and derivatives, flavonoids, glycerophospholipids, benzopyrans, and quinolines. Triatomine feces constitute a rich and varied chemical medium whose constituents are likely to affect <i>T. cruzi</i> development and infectivity. The complexity of the fecal metabolome of triatomines suggests that it may affect triatomine vector competence for specific <i>T. cruzi</i> strains. Knowledge of the chemical environment of <i>T. cruzi</i> in its invertebrate host is likely to generate new ways to understand the factors influencing parasite proliferation as well as methods to control Chagas disease.</p></div

Differences in ion frequencies among triatomine replicates.

<p>Plots are given for all replicate combinations considering the following triatomine pairs: <i>T. infestans</i> vs. <i>R. prolixus</i> (A), <i>T. infestans</i> vs. <i>P. megistus</i> (B) and <i>R. prolixus</i> vs. <i>P. megistus</i> (C). Dots between dashed lines are for the metabolites with small differences among pairs of triatomine species. Dots outside the dashed lines are for the metabolites displaying large differences among pairs of triatomine species (at <i>p</i>≤0.05). For plotting convenience, the scale of the <i>y</i> axis has been limited to the interval −1 to +1. Some pairs exist outside this range (data not shown).</p

Metabolic classes in the uniform core.

<p>Frequency is given in number of hits per metabolic category in the uniform core. Only metabolic classes with 10 or more hits are displayed.</p

Distributions of metabolite rate differences.

<p>Three examples are given for the distributions of these differences among feces of different triatomine species, i.e., <i>T. infestans</i> vs. <i>R. prolixus</i> (A), <i>T. infestans</i> vs. <i>P. megistus</i> (B) and <i>R. prolixus</i> vs. <i>P. megistus</i> (C). The histograms focus on the significant part of the samples in terms of representativeness, but the values were found in a larger interval. In all panels, ∼95% of pair differences are found between −0.15 and +0.15 (n = 2,086).</p

Boolean operations on metabolite rate differences.

<p>Venn diagrams are given for all replicate combinations considering the following triatomine comparisons: <i>R. prolixus</i> vs. <i>T. infestans</i> AND <i>P. megistus</i> (A), <i>T. infestans</i> vs. <i>R. prolixus</i> AND <i>P. megistus</i> (B), <i>P. megistus</i> vs. <i>T. infestans</i> AND <i>R. prolixus</i> (C), and all comparisons above (D).</p

Metabolic classes in the variable core.

<p>Frequency is given in number of hits per metabolic category in the following comparisons: <i>P. megistus</i> vs. <i>T. infestans</i> and <i>R. prolixus</i> (white bars), <i>R. prolixus</i> vs. <i>T. infestans</i> and <i>P. megistus</i> (gray bars), and <i>T. infestans</i> vs. <i>R. prolixus</i> and <i>P. megistus</i> (black bars). Only metabolic classes with 5 or more hits are displayed.</p