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. Mosquitoes are vectors of human pathogens, such as Dengue virus and Malaria parasites, which profoundly affect health worldwide, killing millions of people annually. Recent studies have demonstrated that the presence of bacteria in the gut of mosquitoes is able to antagonize the establishment of pathogens. Therefore, mechanisms with the potential to regulate bacterial growth in the digestive tract of mosquitoes may hamper disease transmission. Here, we show that reactive oxygen species (ROS) are present in the gut epithelia of sugar-fed mosquitoes and are drastically reduced after blood feeding through a mechanism that involves activation of protein kinase C by heme. ROS levels are inversely correlated with the presence of bacteria in the midgut and therefore we investigated if ROS are involved in fighting bacterial infections in the gut. We discovered that mosquitoes producing low levels of ROS challenged with an oral bacterial infection exhibited increased mortality due to intense bacterial proliferation and epithelial cell damage. We propose that reduction in ROS levels after blood feeding is an adaptation to compensate for the ingestion of blood, a pro-oxidant meal. This finding has consequences for the understanding of disease transmission due to the ability of ROS to modulate the gut bacterial levels.

The bloodsucking hemipteran Rhodnius prolixus is a vector of Chagas' disease, which affects 7–8 million people today in Latin America. In contrast to other hematophagous insects, the triatomine gut is compartmentalized into three segments that perform different functions during blood digestion. Here we report analysis of transcriptomes for each of the segments using pyrosequencing technology. Comparison of transcript frequency in digestive libraries with a whole-body library was used to evaluate expression levels. All classes of digestive enzymes were highly expressed, with a predominance of cysteine and aspartic proteinases, the latter showing a significant expansion through gene duplication. Although no protein digestion is known to occur in the anterior midgut (AM), protease transcripts were found, suggesting secretion as pro-enzymes, being possibly activated in the posterior midgut (PM). As expected, genes related to cytoskeleton, protein synthesis apparatus, protein traffic, and secretion were abundantly transcribed. Despite the absence of a chitinous peritrophic membrane in hemipterans - which have instead a lipidic perimicrovillar membrane lining over midgut epithelia - several gut-specific peritrophin transcripts were found, suggesting that these proteins perform functions other than being a structural component of the peritrophic membrane. Among immunity-related transcripts, while lysozymes and lectins were the most highly expressed, several genes belonging to the Toll pathway - found at low levels in the gut of most insects - were identified, contrasting with a low abundance of transcripts from IMD and STAT pathways. Analysis of transcripts related to lipid metabolism indicates that lipids play multiple roles, being a major energy source, a substrate for perimicrovillar membrane formation, and a source for hydrocarbons possibly to produce the wax layer of the hindgut. Transcripts related to amino acid metabolism showed an unanticipated priority for degradation of tyrosine, phenylalanine, and tryptophan. Analysis of transcripts related to signaling pathways suggested a role for MAP kinases, GTPases, and LKBP1/AMP kinases related to control of cell shape and polarity, possibly in connection with regulation of cell survival, response of pathogens and nutrients. Together, our findings present a new view of the triatomine digestive apparatus and will help us understand trypanosome interaction and allow insights into hemipteran metabolic adaptations to a blood-based diet. The bloodsucking bug Rhodnius prolixus is a vector of Chagas' disease, which affects 7–8 million people in Latin America. In contrast to other insects, the digestive tract of Rhodnius has three segments that perform different functions during blood digestion. Here we report analysis of transcriptomes for each of these segments using pyrosequencing technology amounting to several million sequences. Comparison of transcript frequency in digestive libraries with a whole-body library was used to evaluate expression levels, leading to the discovery of several families of enzymes associated with the digestion of proteins, carbohydrates, and lipids, as well as proteins involved in immunity, signal transduction, amino-acid metabolism, and detoxification. Together, our findings present a new view of the triatomine digestive apparatus and will help us understand the mechanism of blood digestion by Rhodnius and its interaction with the agent of Chagas' disease, Trypanosoma cruzi, a parasite that grows within the insect's digestive system.

Background: NADPH Oxidase 5 (Nox5) is a calcium‐sensitive superoxide‐generating Nox. It is present in lower forms and higher mammals, but not in rodents. Nox5 is expressed in vascular cells, but the functional significance remains elusive. Given that contraction is controlled by calcium and reactive oxygen species, both associated with Nox5, we questioned the role of Nox5 in pro‐contractile signaling and vascular function. Methods and Results: Transgenic mice expressing human Nox5 in a vascular smooth muscle cell–specific manner (Nox5 mice) and Rhodnius prolixus , an arthropod model that expresses Nox5 endogenoulsy, were studied. Reactive oxygen species generation was increased systemically and in the vasculature and heart in Nox5 mice. In Nox5‐expressing mice, agonist‐induced vasoconstriction was exaggerated and endothelium‐dependent vasorelaxation was impaired. Vascular structural and mechanical properties were not influenced by Nox5. Vascular contractile responses in Nox5 mice were normalized by N ‐acetylcysteine and inhibitors of calcium channels, calmodulin, and endoplasmic reticulum ryanodine receptors, but not by GKT 137831 (Nox1/4 inhibitor). At the cellular level, vascular changes in Nox5 mice were associated with increased vascular smooth muscle cell [Ca ²⁺ ] _i , increased reactive oxygen species and nitrotyrosine levels, and hyperphosphorylation of pro‐contractile signaling molecules MLC 20 (myosin light chain 20) and MYPT 1 (myosin phosphatase target subunit 1). Blood pressure was similar in wild‐type and Nox5 mice. Nox5 did not amplify angiotensin II effects. In R. prolixus , gastrointestinal smooth muscle contraction was blunted by Nox5 silencing, but not by VAS 2870 (Nox1/2/4 inhibitor). Conclusions: Nox5 is a pro‐contractile Nox isoform important in redox‐sensitive contraction. This involves calcium‐calmodulin and endoplasmic reticulum–regulated mechanisms. Our findings define a novel function for vascular Nox5, linking calcium and reactive oxygen species to the pro‐contractile molecular machinery in vascular smooth muscle cells.

In ticks, the digestion of blood occurs intracellularly and proteolytic digestion of hemoglobin takes place in a dedicated type of lysosome, the digest vesicle, followed by transfer of the heme moiety of hemoglobin to a specialized organelle that accumulates large heme aggregates, called hemosomes. In the present work, we studied the uptake of fluorescent metalloporphyrins, used as heme analogs, and amitraz, one of the most regularly used acaricides to control cattle tick infestations, by Rhipicephalus (Boophilus) microplus midgut cells. Both compounds were taken up by midgut cells in vitro and accumulated inside the hemosomes. Transport of both molecules was sensitive to cyclosporine A (CsA), a well-known inhibitor of ATP binding cassette (ABC) transporters. Rhodamine 123, a fluorescent probe that is also a recognized ABC substrate, was similarly directed to the hemosome in a CsA-sensitive manner. Using an antibody against conserved domain of PgP-1-type ABC transporter, we were able to immunolocalize PgP-1 in the digest vesicle membranes. Comparison between two R. microplus strains that were resistant and susceptible to amitraz revealed that the resistant strain detoxified both amitraz and Sn-Pp IX more efficiently than the susceptible strain, a process that was also sensitive to CsA. A transcript containing an ABC transporter signature exhibited 2.5-fold increased expression in the amitraz-resistant strain when compared with the susceptible strain. RNAi-induced down-regulation of this ABC transporter led to the accumulation of metalloporphyrin in the digestive vacuole, interrupting heme traffic to the hemosome. This evidence further confirms that this transcript codes for a heme transporter. This is the first report of heme transport in a blood-feeding organism. While the primary physiological function of the hemosome is to detoxify heme and attenuate its toxicity, we suggest that the use of this acaricide detoxification pathway by ticks may represent a new molecular mechanism of resistance to pesticides.

Here we report the monitoring of the digestive tract colonization of Rhodnius prolixus by Trypanosoma cruzi using an accurate determination of the parasite load by qPCR coupled with fluorescence and bioluminescence imaging (BLI). These complementary methods revealed critical steps necessary for the parasite population to colonize the insect gut and establish vector infection. qPCR analysis of the parasite load in the insect gut showed several limitations due mainly to the presence of digestive-derived products that are thought to degrade DNA and inhibit further the PCR reaction. We developed a real-time PCR strategy targeting the T. cruzi repetitive satellite DNA sequence using as internal standard for normalization, an exogenous heterologous DNA spiked into insect samples extract, to precisely quantify the parasite load in each segment of the insect gut (anterior midgut, AM, posterior midgut, PM, and hindgut, H). Using combined fluorescence microscopy and BLI imaging as well as qPCR analysis, we showed that during their journey through the insect digestive tract, most of the parasites are lysed in the AM during the first 24 hours independently of the gut microbiota. During this short period, live parasites move through the PM to establish the onset of infection. At days 3–4 post-infection (p.i.), the parasite population begins to colonize the H to reach a climax at day 7 p.i., which is maintained during the next two weeks. Remarkably, the fluctuation of the parasite number in H remains relatively stable over the two weeks after refeeding, while the populations residing in the AM and PM increases slightly and probably constitutes the reservoirs of dividing epimastigotes. These data show that a tuned dynamic control of the population operates in the insect gut to maintain an equilibrium between non-dividing infective trypomastigote forms and dividing epimastigote forms of the parasite, which is crucial for vector competence. Although the key aspects of the T. cruzi life cycle were described more than one century ago, the development and interactions of T. cruzi with its vector are poorly characterized. By dissection of different compartments of the triatomine gut (prototype Rhodnius prolixus) (i.e., AM, PM and H) at regular time intervals, we evaluated trypanosome development within the insect using an accurate qPCR assay. qPCR analysis of trypanosomal colonization and clearance dynamics in real-time were confirmed in vivo using both fluorescence and bioluminescence imaging, which revealed massive parasite lysis during the first 24 hours post-feeding (p.f.). After one week, the parasite succeeded in establishing a resident population in each compartment of the gut, albeit at varying levels. From one week after the onset of infection in the AM and PM, some resident forms agglomerated into rosettes, clustering in close association with the vector tissue and constituting potential parasite reservoirs of the bug. For the first time, we have described a methodology to accurately quantify parasites in the insect gut that would be a useful tool for evaluating the impact of RNAi silencing of insect genes during the course of infection by T. cruzi.

In insects, eggshell hardening involves cross-linking of chorion proteins via their tyrosine residues. This process is catalyzed by peroxidases at the expense of H₂O₂ and confers physical and biological protection to the developing embryo. Here, working with Rhodnius prolixus, the insect vector of Chagas disease, we show that an ovary dual oxidase (Duox), a NADPH oxidase, is the source of the H₂O₂ that supports dityrosine-mediated protein cross-linking and eggshell hardening. RNAi silencing of Duox activity decreased H₂O₂ generation followed by a failure in embryo development caused by a reduced resistance to water loss, which, in turn, caused embryos to dry out following oviposition. Phenotypes of Duox-silenced eggs were reversed by incubation in a water-saturated atmosphere, simultaneous silencing of the Duox and catalase genes, or H₂O₂ injection into the female hemocoel. Taken together, our results show that Duox-generated H₂O₂ fuels egg chorion hardening and that this process plays an essential role during eggshell waterproofing.