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. 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 is in press in Plos Pathogens. We obtained experimental evidence that some African strains of Plasmodium falciparum (NF54 and GB4) are able to evade the mosquito immune system, while others (7G8) 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 used a combination of genetic mapping and linkage group selection to identify a locus in Chr. 13 that confers an African strain of Plasmodium falciparum (GB4) the ability to survive in An. gambiae (L35) females, a strain originally selected to be highly refractory to P. cynomolgy. The QTL encompasses a 171.8 kb region coding for 41 genes. We identified multiple polymorphisms and differences in gene expression between the parental stains that were used to generate a gene candidate priority list. Phenotypic analysis of knockout (KO) lines of our two top candidate genes identified Pfs47 as the gene that allows P. falciparum to evade the immune system of A. gambiae mosquitoes. Pfs47-KO parasites are readily eliminated, but infection can be rescued by disrupting the mosquito complement-like system or by genetic complementation with the NF54 allele of Pfs47. The manuscript describing this work was published in Science. In Africa the Pfs47 gene is highly polymorphic, but in other areas of the world there is a remarkable degree of geographic fixation. This is most extreme in parasites from the New World. We are testing the general hypothesis that Pfs47 has to interact with a mosquito gene to disrupt the immune response and be transmitted more effectively, and that different alleles of Pfs47 work better to evade the immune system of certain mosquito vectors. If this hypothesis is correct, we expect that the different mosquito vectors in Africa will preferentially transmit parasites with certain alleles of Pfs47. The team in Mali is collecting mosquitoes from homes using the """"""""spray-catch"""""""" method and doing ELISA assays to identify Plasmodium-falciparum infected mosquitoes. They are shipping the material from infected mosquitoes to NIH and we are extracting the genomic DNA, sequencing the Pfs47 gene and doing molecular genotyping of the mosquito vector. We are generating transgenic P. falciparum strains with the same genetic background (NF54) that only differ in the allelic variant of Pfs47 they express. Lines with the alleles frequently found in Asia, Africa or the New World are being generated. At NIH, we have established several colonies of anopheline mosquitoes that transmit malaria in different regions of the world (A. albimanus, A. gambiae M-form, A. dirus, A. stephensi). We will also establish an A. aquasalis colony, an important vector in Brazil. We have cloned the TEP1 ortholog genes from each of these mosquito species using degenerate primer PCR. We will infect each mosquito species with parasites expressing different alleles of Pfs47 and the effect of silencing TEP1 on Plasmodium infection will be established. The hypothesis that parasites expressing certain Pfs47 alleles will be detected by the immune system of certain mosquito species, but will be invisible to other mosquito vectors, and the infectivity of the Pfs47 KO line and NF54 WT parasites will be investigated. 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 and we have made substantial progress. HDF appears to consist of a lipocalin that carries a bioactive lipid. We are currently determining the identity of the lipid by LC/MS/MS analysis of the bioactive fractions. Hemocytes synthesize key components of the mosquito complement-like system, but their role in the activation of antiplasmodial responses has not been established. We investigated the effect of activating Toll signaling in hemocytes on Plasmodium survival by transferring hemocytes or cell-free hemolymph from donor mosquitoes in which the suppressor cactus was silenced. These transfers greatly enhanced antiplasmodial immunity, indicating that hemocytes are active players in the activation of the complement-like system, through an effector/effectors regulated by the Toll pathway. A comparative analysis of hemocyte populations between susceptible G3 and the refractory L3-5 Anopheles gambiae mosquito strains did not reveal significant differences under basal conditions or in response to Plasmodium berghei infection. The response of susceptible mosquitoes to different Plasmodium species revealed similar kinetics following infection with P. berghei, P. yoelii or P. falciparum, but the strength of the priming response was stronger in less compatible mosquito-parasite pairs. The Toll, Imd, STAT or We found that the JNK, STAT and Toll pathway are required for hemocytes to respond to HDF, but the IMD pathway is dispensable. Neither the Toll, JNK, STAT nor IMD pathways are required for HDF to be synthesized and released into the mosquito hemolymph. This work was published in the Journal of Innate Immunity. The midgut is the first organ in the mosquito that is invaded by malaria parasites. Although the A. gambiae genome sequencing was completed several years ago, most of the annotation relies on sequence comparisons with other organisms. We carried out Illumina sequence of the midgut transcriptome from mosquitoes infected with P. falciparum, P. berghei or fed with uninfected blood to identify novel transcripts and improve the genome annotation. The analysis of 120 M reads identified 22,889 transcripts. Most of them (76%) represent potentially novel transcripts, while 24% have a complete match to known transcripts. Interestingly, 8,783 (50.5%) of the novel transcripts are intergenic (between two annotated genes) and correspond to 7,745 genes, while the rest represent alternative splicing forms of genes already annotated. A protein coding probability analysis indicates that most of the novel intergenic transcripts (78%) appear to be long non-coding RNAs (lncRNAs). The manuscript describing this work is currently under 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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