We are interested in exploring the interactions between the mosquito (A. gambiae and A. stephensi) immune system and the malaria parasite and to understand how they determine vector competence. Some major areas of interest include: Cell biology and biochemistry of the defense responses of midgut epithelial cells to ookinete invasion: Parasite invasion of susceptible (S) anopheline mosquitoes triggers a series of toxic reactions, such as the generation of nitrogen dioxide (a chemical ?bomb?), so that the ookinete has a limited time-window to escape unharmed. Our studies indicate that inducible peroxidases mediate nitration of mosquito midgut cells in response to Plasmodium invasion. A manuscript describing these results was published. The in vivo responses of mosquito midgut epithelial cells to ookinete invasion in three different vector-parasite combinations, A. aegypti-P. gallinaceum, A. stephensi-P. berghei and A. stephensi-P. gallinaceum, were investigated using enzymatic markers and immunofluorescence stainings. In A. aegypti and A. stephensi, ookinetes traverse the midgut via an intracellular route and inflict irreversible damage to the invaded cells. These two mosquito species differ however in their mechanisms of epithelial repair. A. stephensi removes damaged cells by an actin-mediated budding-off mechanism when invaded by either P. berghei or P. gallinaceum. In A. aegypti, the midgut epithelium is repaired involves the formation of a ?cone-shaped? actin, which closes sequentially, expelling the cellular contents into the midgut lumen as it brings together healthy neighboring cells. Invasion of A. stephensi by P. berghei induced expression of nitric oxide synthase and peroxidase activities, which mediate tyrosine nitration. These two enzymes, however, were not induced in the other two vector-parasite combinations examined and no nitrotyrosine staining could be detected. These studies indicate that the epithelial responses of different mosquito-parasite combinations are not universal. The epithelial responses of the refractory (R) A. gambiae strain to P. berghei invasion were different from those of the susceptible G3 strain in that NOS and peroxidase expression was not induced; no cell protrusion, loss of microvilli, formation of an actin ring, tyrosine nitration nor budding off of the invaded cells were observed. These data indicate that in R mosquitoes P. berghei invasion does not induce apoptosis of midgut cells. When the expression of heme peroxidases was compared between S and R females, a peroxidase that is induced by Plasmodium infection in the S strain was found to be constitutively expressed in the R strain, while a second heme peroxidase that is down-regulated by Plasmodium infection in G3 remained expressed at high levels in R females. Silencing of either one of these genes by systemic dsRNA injection reverted the refractory (R) phenotype, so that healthy developing oocysts were observed 48 h post-infection. The role of reactive oxygen species (ROS) in determining the balance between fecundity, immunity and vectorial capacity in A. gambiae. Hemolymph levels of hydrogen peroxide are significantly lower in sugar-fed super-susceptible females 4a r/r (SS) than in the R strain. In all strains blood feeding caused a significant increase in hydrogen peroxide and levels in fed R females were significantly higher than those of G3 and SS. In all strains, egg production decreased as females aged. When females were blood-fed 8 days post-emergence, the SS strain had significantly higher egg production than G3 and R. Administration of dietary Vitamin C or uric acid, both strong antioxidants, increased egg production in G3 and R females to the same levels as the SS strain. Catalase activity was significantly higher in the midgut of S females at 24 h and in the ovaries 48 h post feeding and the enzyme had a higher pH optimum (9.0) ins S as compared to G3 (8.5). Recent allelic analysis in the three strains revealed that the G3 strain is homozygous for the Cat-w catalase allele (that has a tryptophan at position 2), while the S Strain is homozygous but for the Cat-w catalase allele (that has serine (s) at position 2). The survival of unfed females from the S, G3 and R strains to the same bacterial dose injected into the hemocele was evaluated. The mortality in R strain was significantly lower than in G3, and lower in G3 than in S. To confirm that this inverse relationship between hemolymph levels of hydrogen peroxide and mortality to bacterial challenge could be explained by differences in free radical levels, the effect of dietary administration of vitamin C or uric acid to G3 females was tested. Both antioxidants resulted in a dramatic increase in mortality following bacterial inoculation, illustrating the importance of ROS in anti-bacterial defense responses. Regulation of mRNA expression of genes mediating mosquito immune responses. Completion of the genome sequence revealed the existence of a second member of the STAT family of transcription factors in A. gambiae (we will refer to it as AgSTAT-B). Complete cDNAs for AgSTAT-B and for two suppressors of this pathway in vertebrates, SOCS and PIAS, have been cloned and sequenced. Their protein products were expressed as recombinant proteins in E. coli and used to generate polyclonal antibodies. Both STAT genes, NOS, SOCS and PIAS are expressed in the immuno-responsive A. gambiae Sua 5.1 cell line; and NOS and SOCS are transcriptionally activated in response to bacterial challenge. Silencing of AgSTAT-B by transfecting cells with in vitro synthesized dsRNA resulted in a significant decrease of NOS and SOCS mRNA expression following bacterial challenge, indicating that both transcription factors participate in the regulation of NOS and SOCS expression. NOS and SOCS mRNA expression is induced in response to Plasmodium, and this response is almost completely eliminated (99.7% and 99.6%, respectively) when AgSTAT-B was silenced in the body wall, demonstrating the central role of this transcription factor in the regulation of both genes.