We found that midgut epithelial nitration reactions mediated by heme peroxidase (HPX2) and NADPH oxidase 5 (NOX5) are an important component of antiplasmodial immunity. The HPX2/NOX5 system potentiates NO toxicity and labels ookinetes as they traverse the midgut, making them visible to the mosquito complement-like system. This work was published in Science. We identify the JNK signaling pathway as a key regulator of midgut epithelial nitration by regulating HPX2 and NOX5 expression. This pathway also regulates the basal levels of TEP1 and FBN9 expression in hemocytes. Disruption of the JNK signaling cascade prevents activation of the Anopheles gambiae complement-like immune system. The manuscript describing this work in preparation. We identified an epithelial serine protease (ESP) that is required for Plasmodium parasites to efficiently invade midgut epithelial cells. Midgut invasion by ookinetes and sporozoite invasion of the salivary gland induces SP30 expression. Furthermore, SP30 silencing significantly decreases the number of parasites that invade these organs. ESP protein is localized in the submicrovillar region of midgut cells and the basal side of salivary gland epithelial cells, the surfaces that parasites interact with during invasion. In the midgut, ESP silencing prevents the induction of Gelsolin, a protein involved in modulating the actin cytoskeleton that is known to also be required for midgut invasion. This work was published in Plos One. Previous studies showed that Anopheles gambiae L3-5 females, which are refractory (R) to Plasmodium infection, express higher levels of genes involved in redox-metabolism and mitochondrial respiration than susceptible (S) G3 females. Our studies revealed that R females have reduced longevity, faster utilization of lipid reserves, impaired mitochondrial state-3 respiration, increased rate of mitochondrial electron leak and higher expression levels of several glycolytic enzyme genes. Furthemore, reducing state-3 respiration by silencing the adenine translocator increased hydrogen peroxide generation and reduced susceptibility to Plasmodium infection. This work was published in Insect Biochem. Mol. Biol. We found that a novel member of the mitochondrial transporter protein family, Anopheles gambiae mitochondrial carrier 1 (AgMC1), is required to maintain mitochondrial membrane potential in mosquito midgut cells. AgMC1 silencing reduces mitochondrial membrane potential, resulting in increased proton-leak and uncoupling of oxidative phosphorylation. These metabolic changes reduce midgut ROS generation and increase A. gambiae susceptibility to Plasmodium infection. We provide direct experimental evidence indicating that ROS derived from mitochondria can modulate mosquito epithelial responses to Plasmodium infection. This work was published in Plos One. We found that adult mosquito females challenged with Plasmodium respond more efficiently to subsequent challenges and that the transfer of cell-free hemolymph from challenged mosquitoes to newly emerged females induces hemocyte differentiation and confers increased resistance to Plasmodium infection. We are currently in the process of characterizing this hemocyte differentiation factor (HDF) using a biochemical approach. We found that the JNK, STAT and Toll pathway are required for hemocytes to respond to HDF, but the IMD pathway is dispensable. The Toll pathways is not require for the synthesis of HDF and the participation of the other pathways is under investigation. We obtained experimental evidence that some African strain of Plasmodium falciparum are able to evade the mosquito immune system, while other are readily recognized and eliminated. This effect is parasite-autonomous, because, in co-infection experiments, the recognition (or lack of) of one strain does not affect the outcome of the other. We also identified important genetic differences between the melanization responses to P. berghei and P. falciparum infection. This work was publishes in PNAS. We identified a QTL in Chr. 13 that confers an African strain of Plasmodium falciparum (GB4) the ability to survive in An. gambiae (L35) females. In contrast, 98-100% of P. falciparum 7G8 parasites (Brazilian strain) are detected by the mosquito immune system and killed. The QTL encompasses a 171.8 kb region coding for 41 genes. We confirmed that QTL regions using linkage group selection, identified multiple polymorphisms and differences in gene expression. Phenotypic analysis of knockout lines for candidate genes made it possible to identify the gene that allows P. falciparum to evade the immune system of A. gambiae. The manuscript describing this work is in preparation.

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Molina-Cruz, Alvaro; Barillas-Mury, Carolina (2016) Mosquito vectors of ape malarias: Another piece of the puzzle. Proc Natl Acad Sci U S A 113:5153-4
Orfano, Alessandra S; Nacif-Pimenta, Rafael; Duarte, Ana P M et al. (2016) Species-specific escape of Plasmodium sporozoites from oocysts of avian, rodent, and human malarial parasites. Malar J 15:394
Ramirez, Jose Luis; de Almeida Oliveira, Giselle; Calvo, Eric et al. (2015) A mosquito lipoxin/lipocalin complex mediates innate immune priming in Anopheles gambiae. Nat Commun 6:7403
Molina-Cruz, Alvaro; Canepa, Gaspar E; Kamath, Nitin et al. (2015) Plasmodium evasion of mosquito immunity and global malaria transmission: The lock-and-key theory. Proc Natl Acad Sci U S A 112:15178-83
Ramphul, Urvashi N; Garver, Lindsey S; Molina-Cruz, Alvaro et al. (2015) Plasmodium falciparum evades mosquito immunity by disrupting JNK-mediated apoptosis of invaded midgut cells. Proc Natl Acad Sci U S A 112:1273-80
Herrera, Raul; Anderson, Charles; Kumar, Krishan et al. (2015) Reversible conformational change in the Plasmodium falciparum circumsporozoite protein masks its adhesion domains. Infect Immun :
Smith, Ryan C; Barillas-Mury, Carolina; Jacobs-Lorena, Marcelo (2015) Hemocyte differentiation mediates the mosquito late-phase immune response against Plasmodium in Anopheles gambiae. Proc Natl Acad Sci U S A 112:E3412-20
Parra, Marcela; Liu, Xia; Derrick, Steven C et al. (2015) Co-expression of Interleukin-15 Enhances the Protective Immune Responses Induced by Immunization with a Murine Malaria MVA-Based Vaccine Encoding the Circumsporozoite Protein. PLoS One 10:e0141141
Padron, Alejandro; Molina-Cruz, Alvaro; Quinones, Mariam et al. (2014) In depth annotation of the Anopheles gambiae mosquito midgut transcriptome. BMC Genomics 15:636
Waisberg, Michael; Molina-Cruz, Alvaro; Mizurini, Daniella M et al. (2014) Plasmodium falciparum infection induces expression of a mosquito salivary protein (Agaphelin) that targets neutrophil function and inhibits thrombosis without impairing hemostasis. PLoS Pathog 10:e1004338

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