Modulating Autophagy in Mosquitoes to Understand P. vivax Infection

The transcriptome associated to P. vivax infection revealed a variety of transcripts that play a key role in autophagy. In order to evaluate the effect of the autophagy process in the outcome of infection, we inoculated mosquitoes with wortmannin (an inhibitor of phosphatidylinositol 3-kinase DPI3K) and spermidine (an autophagy activator) [36 (link), 37 (link)]. Three- to four-day-old female mosquitoes were cold-anesthetized and inoculated intrathoraxically with 69 nl of a 5 μM and 0.05 μM solution of wortmannin (Merck, Darmstadt, Germany) or with the same volume of H₂O Ultra Pure and with 69 nl of a 100 μM solution of spermidine (Sigma) or DMSO (0.05%) using a Nanoject micro-injector (Drummond Scientific, Pennsylvania, USA). Twenty-four hours after injection with the solutions, the mosquitoes were fed with a P. vivax-infected blood meal as described above. Three independent biological replicates were performed for each experiment. Mosquitoes were dissected 18–24 h after feeding; batches of 20–30 midguts were dissected in cold DEPC-treated phosphate-buffered saline (PBS) and processed for RNA preparation and cDNA synthesis using the same protocols mentioned above. Mosquito midguts were also collected on the 8th day post-infection to determine the prevalence and intensity of infection.