Blood Meal-Derived Heme Decreases ROS Levels in the Midgut of Aedes aegypti and Allows Proliferation of Intestinal Microbiota

The presence of bacteria in the midgut of mosquitoes antagonizes infectious agents, such as Dengue and Plasmodium, acting as a negative factor in the vectorial competence of the mosquito. Therefore, knowledge of the molecular mechanisms involved in the control of midgut microbiota could help in the development of new tools to reduce transmission. We hypothesized that toxic reactive oxygen species (ROS) generated by epithelial cells control bacterial growth in the midgut of Aedes aegypti, the vector of Yellow fever and Dengue viruses. We show that ROS are continuously present in the midgut of sugar-fed (SF) mosquitoes and a blood-meal immediately decreased ROS through a mechanism involving heme-mediated activation of PKC. This event occurred in parallel with an expansion of gut bacteria. Treatment of sugar-fed mosquitoes with increased concentrations of heme led to a dose dependent decrease in ROS levels and a consequent increase in midgut endogenous bacteria. In addition, gene silencing of dual oxidase (Duox) reduced ROS levels and also increased gut flora. Using a model of bacterial oral infection in the gut, we show that the absence of ROS resulted in decreased mosquito resistance to infection, increased midgut epithelial damage, transcriptional modulation of immune-related genes and mortality. As heme is a pro-oxidant molecule released in large amounts upon hemoglobin degradation, oxidative killing of bacteria in the gut would represent a burden to the insect, thereby creating an extra oxidative challenge to the mosquito. We propose that a controlled decrease in ROS levels in the midgut of Aedes aegypti is an adaptation to compensate for the ingestion of heme

Evolutionary origin and function of NOX4-art, an arthropod specific NADPH oxidase

Abstract Background NADPH oxidases (NOX) are ROS producing enzymes that perform essential roles in cell physiology, including cell signaling and antimicrobial defense. This gene family is present in most eukaryotes, suggesting a common ancestor. To date, only a limited number of phylogenetic studies of metazoan NOXes have been performed, with few arthropod genes. In arthropods, only NOX5 and DUOX genes have been found and a gene called NOXm was found in mosquitoes but its origin and function has not been examined. In this study, we analyzed the evolution of this gene family in arthropods. A thorough search of genomes and transcriptomes was performed enabling us to browse most branches of arthropod phylogeny. Results We have found that the subfamilies NOX5 and DUOX are present in all arthropod groups. We also show that a NOX gene, closely related to NOX4 and previously found only in mosquitoes (NOXm), can also be found in other taxonomic groups, leading us to rename it as NOX4-art. Although the accessory protein p22-phox, essential for NOX1-4 activation, was not found in any of the arthropods studied, NOX4-art of Aedes aegypti encodes an active protein that produces H2O2. Although NOX4-art has been lost in a number of arthropod lineages, it has all the domains and many signature residues and motifs necessary for ROS production and, when silenced, H2O2 production is considerably diminished in A. aegypti cells. Conclusions Combining all bioinformatic analyses and laboratory work we have reached interesting conclusions regarding arthropod NOX gene family evolution. NOX5 and DUOX are present in all arthropod lineages but it seems that a NOX2-like gene was lost in the ancestral lineage leading to Ecdysozoa. The NOX4-art gene originated from a NOX4-like ancestor and is functional. Although no p22-phox was observed in arthropods, there was no evidence of neo-functionalization and this gene probably produces H2O2 as in other metazoan NOX4 genes. Although functional and present in the genomes of many species, NOX4-art was lost in a number of arthropod lineages

Heme crystallization in the midgut of triatomine insects

Submitted by Martha Martínez Silveira ([email protected]) on 2015-05-20T17:50:01Z No. of bitstreams: 1 Oliveira MF Heme crystalization in the midgut......pdf: 470893 bytes, checksum: 07f8324e561a2a3503de46cea2de522c (MD5)Approved for entry into archive by Martha Martínez Silveira ([email protected]) on 2015-05-20T18:02:53Z (GMT) No. of bitstreams: 1 Oliveira MF Heme crystalization in the midgut......pdf: 470893 bytes, checksum: 07f8324e561a2a3503de46cea2de522c (MD5)Made available in DSpace on 2015-05-20T18:02:53Z (GMT). No. of bitstreams: 1 Oliveira MF Heme crystalization in the midgut......pdf: 470893 bytes, checksum: 07f8324e561a2a3503de46cea2de522c (MD5) Previous issue date: 2007Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica. Rio de Janeiro, RJ, BrasilUniversidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica. Rio de Janeiro, RJ, BrasilPetrobrás. CENPES. Setor de Química. Rio de Janeiro, RJ, BrasilUniversidade Estadual do Norte Fluminense. Centro de Biociências e Biotecnologia. Campos de Goytacazes, RJ, BrasilUniversidade Estadual do Norte Fluminense. Centro de Biociências e Biotecnologia. Campos de Goytacazes, RJ, BrasilUniversidade Estadual do Norte Fluminense. Centro de Biociências e Biotecnologia. Campos de Goytacazes, RJ, BrasilFundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, BrasilUniversidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica. Rio de Janeiro, RJ, BrasilHemozoin (Hz) is a heme crystal produced by several blood-feeding organisms in order to detoxify free heme released upon hemoglobin (Hb) digestion. Here we show that heme crystallization also occurs in three species of triatomine insects. Ultraviolet-visible and infrared light absorption spectra of insoluble pigments isolated from the midgut of three triatomine species Triatoma infestans, Dipetalogaster maximus and Panstrongylus megistus indicated that all produce Hz. Morphological analysis of T. infestans and D. maximus midguts revealed the close association of Hz crystals to perimicrovillar membranes and also as multicrystalline assemblies, forming nearly spherical structures. Heme crystallization was promoted by isolated perimicrovillar membranes from all three species of triatomine bugs in vitro in heat-sensitive reactions. In conclusion, the data presented here indicate that Hz formation is an ancestral adaptation of Triatominae to a blood-sucking habit and that the presence of perimicrovillar membranes plays a central role in this process. © 2007 Elsevier Inc. All rights reserve

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

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

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

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

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

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