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Aedes aegypti mosquitoes transmit arboviruses of important global health impact, and their intestinal microbiota can influence vector competence by stimulating the innate immune system. Midgut epithelial cells also produce toxic reactive oxygen species (ROS) by dual oxidases (DUOXs) that are essential players in insect immunity. Strigomonas culicis is a monoxenous trypanosomatid that naturally inhabits mosquitoes; it hosts an endosymbiotic bacterium that completes essential biosynthetic pathways of the parasite and influences its oxidative metabolism. Our group previously showed that S. culicis hydrogen peroxide (H2O2)-resistant (WTR) strain is more infectious to A. aegypti mosquitoes than the wild-type (WT) strain. Here, we investigated the influence of both strains on the midgut oxidative environment and the effect of infection on mosquito fitness and immunity. WT stimulated the production of superoxide by mitochondrial metabolism of midgut epithelial cells after 4 days post-infection, while WTR exacerbated H2O2 production mediated by increased DUOX activity and impairment of antioxidant system. The infection with both strains also disrupted the fecundity and fertility of the females, with a greater impact on reproductive fitness of WTR-infected mosquitoes. The presence of these parasites induced specific transcriptional modulation of immune-related genes, such as attacin and defensin A during WTR infection (11.8- and 6.4-fold, respectively) and defensin C in WT infection (7.1-fold). Thus, we propose that A. aegypti oxidative response starts in early infection time and does not affect the survival of the H2O2-resistant strain, which has a more efficient antioxidant system. Our data provide new biological aspects of A. aegypti-S. culicis relationship that can be used later in alternative vector control strategies.

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Low levels of reactive oxygen species (ROS) are now recognized as essential players in cell signaling. Here, we studied the role of two conserved enzymes involved in redox regulation that play a critical role in the control of ROS in the digestive physiology of a blood-sucking insect, the kissing bug Rhodnius prolixus. RNAi-mediated silencing of RpNOX5 and RpXDH induced early mortality in adult females after a blood meal. Recently, a role for RpNOX5 in gut motility was reported, and here, we show that midgut peristalsis is also under the control of RpXDH. Together with impaired peristalsis, silencing either genes impaired egg production and hemoglobin digestion, and decreased hemolymph urate titers. Ultrastructurally, the silencing of RpNOX5 or RpXDH affected midgut cells, changing the cells of blood-fed insects to a phenotype resembling the cells of unfed insects, suggesting that these genes work together in the control of blood digestion. Injection of either allopurinol (an XDH inhibitor) or uricase recapitulated the gene silencing effects, suggesting that urate itself is involved in the control of blood digestion. The silencing of each of these genes influenced the expression of the other gene in a complex way both in the unfed state and after a blood meal, revealing signaling crosstalk between them that influences redox metabolism and nitrogen excretion and plays a central role in the control of digestive physiology.

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The termite gut is an efficient decomposer of polyphenol-rich diets, such as lignocellulosic biomasses, and it has been proposed that non-enzymatic oxidative mechanisms could be involved with the digestive process in these animals. However, oxidant levels are completely unknown in termites, as well as protective mechanisms against oxidative damage to the termite gut and its microbiota. As the first step in investigating the role oxidants plays in termite gut physiology, this work presents oxidant levels, antioxidant enzymatic defenses, cell renewal and microbiota abundance along the litter-feeding termite Cornitermes cumulans gut compartments (foregut, midgut, mixed segment and hindgut p1, p3, p4, and p5 segments) and salivary glands. The results show variable levels of oxidants along the C. cumulans gut, the production of antioxidant enzymes, gut cell renewal as potential defenses against oxidative injuries and the profile of microbiota distribution (being predominantly inverse to oxidant levels). In this fashion, the oxidative challenges imposed by polyphenol-rich diet seem to be circumvented by the C. cumulans gut, ensuring efficiency of the digestive process together with preservation of tissue homoeostasis and microbiota growth. These results present new insights into the physicochemical properties of the gut in a litter-feeding termite, expanding our view in relation to termites' digestive physiology.

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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 Trypanosoma cruzi, 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 T. cruzi, the physiological relevance of hemozoin for these organisms remains unknown. Here, we demonstrate that selective blockage of heme crystallization in vivo 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 T. cruzi parasite counts in R. prolixus 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 T. cruzi life-cycle. Thus, targeting heme crystallization in insect vectors represents an innovative way for Chagas disease control, by reducing simultaneously triatomine reproduction and T. cruzi transmission.

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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.

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