How do insects fight pathogens? Molecular and functional characterization of the immune deficiency pathway in Rhodnius prolixus, a vector of parasites that cause Chagas disease

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(Thesis) Ph.D.
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Rhodnius prolixus is a triatomine insect that has been used for >80 years as a model organism to study many aspects of insect physiology. It is also a vector of Trypanosoma cruzi, the causal agent of human Chagas disease. Triatomines interact through their immune systems with myriad microorganisms including trypanosomes and other pathogens that must be tolerated or eliminated, and beneficial symbionts that must be nourished. Pioneering studies on insect immunity were described in holometabolous insects, and it was assumed that that the innate immune system was conserved in all insects. The publication of the R. prolixus genome, however, revealed that one of the main immune pathways, the Immune-deficiency pathway (IMD), was fragmented, with many missing genes, and probably was non-functional. To investigate whether the IMD pathway exists and is functional in R. prolixus we used a combination of bioinformatics and molecular analyses. We used sequence homology searches, 3D protein modeling, phylogenetic analyses, and RNA sequencing to identify most of the missing genes of the IMD pathway R. prolixus. We used RNA Interference (RNAi) mediated silencing, to functionally characterize the transcription factor Relish, the negative regulator Caspar, and the Peptidoglycan Recognition Receptors, and confirmed that these genes regulate the production of antimicrobial peptides in the fat body. We used RNA-sequencing to generate a transcriptome of all genes up- or down regulated in the fat body tissues after infections with bacteria. We demonstrate that the IMD pathway in R. prolixus responds principally to Gram-negative bacteria and to a lesser degree to Gram-positive bacteria, which deviates from the classical holometabolous insect model. We also found an IMD candidate gene which, in other insects, is essential for the IMD pathway to function. Our RNAi approaches, however, failed to confirm its role in immunity. Our results confirm that the IMD pathway is present and functional in R. prolixus, albeit with important variations compared with other insects. Altogether, these findings have added to the foundation of knowledge on the immune systems of triatomines and other hemimetabolous insects, allowing us to better understand the evolution of insect immunity. This knowledge will allow us to explore in more detail the interactions among triatomines, trypanosomes, and beneficial symbionts with the hope of developing new control strategies for Chagas disease
414 pages.
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Thesis advisor: Lowenberger, Carl
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