Oxidative stree management is essential for anopheles mosquito survival post plasmodium infected blood meal ingestion

A Dissertation Submitted in Partial Fulfilment of the Requirements for the Degree of Doctor of Philosophy in Life Sciences of the Nelson Mandela African Institution of Science and TechnologyAnopheles mosquitoes like other dipterans lack the flavoenzyme glutathione reductase (GR) of the GSH pathway and instead utilize the Thioredoxin (Trx) system for management of oxidative stress. Anopheles gambiae (An. gambiae) and Anopheles stephensi (An. stephensi) mosquitoes have been shown to regulate genes and proteins of the Trx system to protect midgut epithelial cells against reactive oxygen/nitrogen species (ROS/RNS) associated with Plasmodium berghei (P. berghei) infection. However, this mosquito vector-parasite combination is not natural and may not necessarily reflect human malaria transmission biology in the field. Despite its importance, a complete understanding of the Trx pathway at the molecular level is missing. Mosquito feeding assays were used to examine the Trx response pathway following midgut exposure to ROS/RNS, by measuring first the protein expression level of Thioredoxin-1 (AgTrx1) post exposure to tert-Butyl hydroperoxide (tBHP) by quantitative immunoblot analysis. This was followed by measuring the global proteomic response to Paraquat (Pqt) and tBHP exposure. The proteomic response was then compared to a spectrum of Trx- and GSH-dependant transcripts 24 hours post-infected bloodmeal ingestion in the more natural vector-parasite combination of An. gambiae-Plasmodium falciparum (P. falciparum) to assess for: (a) concordance between protein and transcript under different oxidative conditions and (b) similarity to the unnatural vectorparasite (An. gambiae/An. stephensi-P. berghei). It was observed that protein levels of AgTrx-1 remained unchanged in midgut epithelial cells exposed to different concentrations of tBHP. Moreover, proteomics profiles of midgut epithelial cells under tBHP-and Pqt-induced oxidative stress showed cells that are undergoing redox regulation through ribosomal/nucleolar and ERstress responses, respectively. This response is contrary to the canonical antioxidant response previously described. Furthermore, transcript data showed an absence of significant upregulation in the Trx- and GSH-dependent genes. This is consistent with the concept that P. falciparum does not induce marked midgut destruction in An. gambiae, therefore its invasion process is associated with reduced oxidative stress. The ribosomal/nucleolar and ER stress responses to oxidative stress suggest additional response mechanisms to the canonical antioxidant responses. These additional responses could be translated to develop strategies that could lead to unmanageable levels of ROS/RNS exposure to the parasite in the midgut, yet still allowing the mosquito to survive the dysregulation. This would lead to a stop in parasite development leading to blocking of transmission