The role of LuloPAT amino acid/proton symporters in midgut alkalinization in the sandfly Lutzomyia longipalpis (Diptera - Psychodidae).

In Lutzomyia longipalpis females, which are the main vectors of Leishmania infantum in the Americas, hematophagy is crucial for ovary development. The control of pH in the midgut during blood digestion is important to the functioning of the digestive enzymes, which release amino acids in the luminal compartment that are then transported through the enterocytes to the hemolymph for delivery to the ovary and other organs. In the present work, we investigated transport systems known as LuloPATs that are present in the midgut of L. longipalpis but not in other organs. These transporters achieve symport of amino acids with H+ ions, and one of them (LuloPAT1) is orthologous to a transporter described in Aedes aegypti. According to our results, the transcription levels of LuloPAT1 increased significantly immediately after a blood meal. Based on the variation of the fluorescence of fluorescein with the pH of the medium, we developed a technique that shows the acidification of the cytoplasm of gut cells when amino acids are cotransported with H+ from the lumen into the enterocytes. In our experiments, the midguts of the sandflies were dissected and opened longitudinally so that added amino acids could enter the enterocytes via the lumen (PAT carriers are apical). LuloPAT1 transporters are part of a complex of mechanisms that act synergistically to promote gut alkalinization as soon as blood intake by the vector occurs. In dissected but not longitudinally opened midguts, added amino acids could only enter through the basolateral region of enterocytes. However, alkalinization of the lumen was observed because the entry of some amino acids into the cytoplasm of enterocytes triggers a luminal alkalinization mechanism independent of LuloPATs. These findings provide new perspectives that will enable the characterization of the set of signaling pathways involved in pH regulation within the L. longipalpis midgut.

Colonization of Rhodnius prolixus gut by Trypanosoma cruzi involves an extensive parasite killing.

Trypanosoma cruzi, the etiological agent of Chagas disease, is ingested by triatomines during their bloodmeal on an infected mammal. Aiming to investigate the development and differentiation of T. cruzi inside the intestinal tract of Rhodnius prolixus at the beginning of infection we fed insects with cultured epimastigotes and blood trypomastigotes from infected mice to determine the amount of recovered parasites after ingestion. Approximately 20% of the ingested parasites was found in the insect anterior midgut (AM) 3 h after feeding. Interestingly, a significant reduction (80%) in the numbers of trypomastigotes was observed after 24 h of infection suggesting that parasites were killed in the AM. Moreover, few parasites were found in that intestinal portion after 96 h of infection. The evaluation of the numbers of parasites in the posterior midgut (PM) at the same periods showed a reduced parasite load, indicating that parasites were not moving from the AM. Additionally, incubation of blood trypomastigotes with extracts from R. prolixus AMs revealed that components of this tissue could induce significant death of T. cruzi. Finally, we observed that differentiation from trypomastigotes to epimastigotes is not completed in the AM; instead we suggest that trypomastigotes change to intermediary forms before their migration to the PM, where differentiation to epimastigotes takes place. The present work clarifies controversial points concerning T. cruzi development in insect vector, showing that parasite suffers a drastic decrease in population size before epimastigonesis accomplishment in PM.