Reverse genetics of mosquito systemic immunity

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DESCRIPTION (provided by applicant): The long-term goal of this application is to elucidate the molecular mechanisms regulating immune responses in the mosquito Aedes aegypti. In this application, reverse-genetics tools will be developed to study these mechanisms. We have already generated a transgenic, dominant-negative Relish mosquito strain (RMID), which after taking a bloodmeal becomes immune deficient to Gram-negative bacteria (Shin et al., 2003). The latter accomplishment has opened the door to utilization of the heritable reverse-genetics approach in studying mosquito immunity. The RMID transgenic mosquitoes provide an invaluable tool to assess the properties of AMPs in various genetic crosses involving transgenic knock-outs of immune pathways. We will investigate the role of the IMD/Relish pathway in antibacterial and anti-parasite immunity. We will test the hypothesis that Relish plays a key role in humoral immunity in mosquitoes: the effect of Relish knock-out and bloodmeal-activated, over-expressed Relish in transgenic mosquitoes on immune responses will be characterized in detail. We will examine the hypothesis that Dorsal, which is the key transcription factor in the Toll pathway, has a dual role in systemic immunity: (1) activating anti-bacterial and anti-fungal effectors and (2) being responsible for the production of regulatory molecules, such as cytokines, serine pro-proteases and their inhibitors (serpins). Stable, dominant-negative and bloodmeal-activated, over-expressed Dorsal Aedes strains will be generated. Interaction between IMD/Relish and Toll pathways will be investigated through establishing genetically stable Relish/Dorsal hybrid strains. High-throughput gene expression analyses, utilizing cDNA microarrays, will be employed to determine the repertoire of effector and signaling genes being regulated by the two studied pathways those that are induced by infection with bacterial, fungal and Plasmodium pathogens. The genetically stable knock-out and over-expression transgenic strains with altered major immune pathways, in combination with infections, will be employed in microarray assays to establish the regulatory links among pathogen-pathway-effector/signaling genes. Utilization of genetically stable dominant-negative and over-expression strains in combination with a genomic analysis of immune responses is expected to yield information about the key genes regulated by these pathways.