Interação triatomíneos-Trypanosoma cruzi: desenvolvimento de diferentes DTUs, papel das vesículas extracelulares e tentativa de hibridização

Abstract

This project investigated some aspects of Trypanosoma cruzi interaction with Triatoma infestans and Rhodnius prolixus. It was investigated: the role of the triatominae as as a biological barrier for different genetic clones (DM28c-TcI, YuYu-TcII, Bug2145cl10-TcV and CL Brener-TcVI), the colonization dynamics of the initial colonization process of gut by the parasites, the role of extracellular vesicles (EVs) and finally, the occurrence of hybridization in the insect vector. We performed in vivo experiments using artificial feeders containing inactivated rabbit blood and cultured epimastigotes. For the biological barrier experiments, the one triatomine specie was artificially infected with four T. cruzi clones. The insects were followed for 49 days. All clones developed metacyclic forms in both vectors. However, Bug and YuYu clones reached higher densities in the digestive tract. For the initial colonization dynamics of the digestive tract of T. Infestans was used to the strains DM28c and Bug. In the initial events after infective feeding (3h), there was a decrease in the number of parasites in the anterior midgut (AM) in all lineages and insects used. After 72h, there was still presence of AM parasites in the model T. infestans infested with DM28c, but for Bug there were no parasites. Regarding the anterior midgut plus posterior or rectum intestine (PM + R) the parasites were only found for all models and lineages after 72 h. Regarding T. cruzi EVs, they were obtained, purified and artificially offered with rabbit blood on the 1st day of feeding. On the 2nd day, the insects were artificially fed with cultured epimastigotes from the same clone. The insects were followed for 49 days. Pre-feeding with EVs delayed migration of parasites to the posterior parts of the gut in R. prolixus, but not in T. infestans. To evaluate the occurrence of hybridization, the two clones originally isolated from T. infestans that reached higher densities (Bug and YuYu) were used. The insects were infected with T. cruzi Bug and YuYu transfected with fluorescent proteins RFP and GFP, respectively. The insects were followed for 28 days, fed and their diuresis soon after feeding were collected in LIT medium containing the two selection antibiotics (G418 and Hygromycin B). The diuresis resulting from an insect generated viable parasites and by PCR it was not possible to confirm the existence of a hybrid, because the isolate was a TcV equal to its parent. Our preliminary findings indicate that the digestive tubes of the species T. infestans presented a selection paper to different lineages of T. cruzi, therefore, they have the ability to select populations and clones of T. cruzi, acting as biological barrier. Different densities of total parasites were observed and all lineages were able to perform metacylogenesis. Regarding the initial dynamics of infection there is a difenreça in the development of Dm28c in relation to Bug in T. infestans. Bug strain development varied according to the vector and EVs presence, whose migration and colonization to the posterior parts of the gut was delayed. Our preliminary findings did not confirm the occurrence of hybridization within T. infestans.