243 million malaria cases and 863,000 attributed deaths were reported globally in 2009. To be transmitted from one person to another, the malaria parasite (Plasmodium spp.) has to complete an elaborate developmental program in the mosquito and survive the attacks from mosquito innate immune system. Our long term goal is to discover mosquito microRNAs that are involved in anti-Plasmodium defense, elucidate the molecular functions of these microRNAs, and to use this knowledge to aid future efforts to control malaria transmission. MicroRNAs are small endogenous regulatory molecules in most eukaryotes. In animals, mature microRNAs are integrated into a microRNA-induced silencing complex (miRISC) and associated with the 3' untranslated regions of specific target mRNAs to suppress gene expression either through inhibition of translation or through mRNA degradation. In Anopheles gambiae mosquitoes, levels of some microRNAs are markedly affected in the midgut by Plasmodium infection. RNAi-mediated silencing of either Dicer-1 or Argonaute-1, key components of the microRNA pathway, has been shown to enhance parasite survival. We therefore hypothesize that microRNAs play an important role in modulating mosquito defense response to malaria parasites. To test this hypothesis, we need to find mosquito mRNAs that are specifically regulated by microRNAs only after mosquitoes are exposed to malaria parasites in a blood meal. microRNA target prediction by computational approaches alone is generally hampered by high false-positive rates. We will use the HITS-CLIP (high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation) method instead to capture and identify both the microRNAs and target mRNAs bound by Argonaute-1 in miRISC. This will reveal precise target sites across all messenger RNAs expressed in the midgut. The specific aims of this project are to: (1) Determine Anopheles gambiae microRNAs that are enriched in miRISC during Plasmodium falciparum invasion. (2) Identify mosquito messenger RNAs that are selectively regulated by microRNAs in response to Plasmodium challenge. Our proposed studies address a serious gap in the understanding of microRNA functions in the mosquito-Plasmodium interactions, and may provide novel molecular targets for blocking malaria transmission.