Molecular characterization of two aquaporins putatively involved in water, urea, and glycerol transport in the midgut of Rhodnius prolixus

Abstract

Rhodnius prolixus blood meal ingestion activates a cascade of hormonally coordinated events involving different tissues to eliminate excess water and sodium chloride from the blood. This postprandial diuresis stimulates defecation/urination, thus reducing its body weight, preventing them from predation, and allowing blood digestion. When R. prolixus is infected, the parasite Trypanosoma cruzi exits with the feces, eventually infecting the host and causing Chagas' disease. Two primary tissues are involved in this process, the anterior midgut, which absorbs water and sodium chloride, and the Malpighian tubules that secrete them. After reducing the blood volume in the anterior region, digestion of macromolecules and absorption of nutrients occur mainly in the middle and posterior midgut. Preliminary results in the last FAPESP report showed the specific expression of the genes RpPrip and RpEglp1 in the midgut, with the highest expression in the anterior and posterior regions, respectively. Furthermore, bioinformatic and phylogenetic analyses of the encoded amino acid sequences suggest that these proteins could act as water and urea, and water, urea, and glycerol transporters, respectively, indicating the potential role of RpPrip in postprandial diuresis of the anterior region and of RpEglp1 in glycerol transport in the posterior midgut of R. prolixus. The current project will characterize the biophysical properties of RpPrip and RpEglp1 genes through the ectopic expression of corresponding cRNAs in Xenopus laevis oocytes to confirm their role in R. prolixus midgut. Water and solute permeability of RpPrip and RpEglp1 will be tested by oocyte swelling experiments. In both cases, the key amino acid residues determining channel function will be identified using site-directed mutagenesis. These data will complement current in vivo experiments with inhibitors, immunostaining, and gene expression quantification, designed to uncover the physiological role of RpPrip during postprandial diuresis and RpEglp1 in glycerol absorption. (AU)