The mosquito Aedes aegypti ingests large amount of vertebrate blood in a single meal and the hydrolysis of hemoglobin inside its digestive tract releases huge amounts of free heme, which is pro-oxidant and can lead to an oxidative stress situation through an increase in the production of reactive oxigen species (ROS). To counteract this scenario hematophagous insects posses a great number of antioxidat defenses that make possible its survivor.
Besides the toxic effects of ROS it has been shown that these molecules have important physiological roles in a wide range of processes like cellular proliferation, inflammation and microbial killing. Little is known about the role of ROS in insect physiology, although a few recent papers have demonstrated that theses molecules might be involved in the vectorial capacity of mosquitoes. Concerning this background the aim of our work is to characterize the production of ROS in the midgut of Aedes aegypti.
Females were fed with sugar ou blood and their midguts were dissected in PBS and incubated in L-15-5% FBS medium supplemented with CM-H2DCFDA (Molecular Probes), a redox sensitive fluorofur. After at least 30 minutes of incubation the midguts were visualized using an epifluorescence or confocal microscope.
Epifluorescence microscopy results suggested that ROS production is greater in sugar-fed mosquitos and decreases immediately after a blood or plasma meal to background levels until 24 hours after the meal.
The same approach using confocal microscopy revealed another set of information. In sugar-fed females ROS production is localized in two groups of cells with distinct patterns. The majotiry of cells produces ROS in the membrane, following a honeycomb profile. The other small group of cells produces ROS in the entire cytoplasm. Another important conclusion is that the gut lumen is filled with ROS. In blood-fed mosquitoes ROS production is localized inside granules that might be mitochondria distributed throughout the cytoplasm.
Finally, midguts from sugar-fed females were incubated in the presençe of inhibitors of NADPH Oxidase system, apocynin and diphenylene iodonium (DPI). Both inhibitors were able to block the production of ROS in the midgut. Our working hypothesis is that NADPH Oxidase produces ROS that is released in the midgut lumem and helps to control the gut microbiota.