Hemocyte Transcriptome and Immunity in Aedes aegypti

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The mosquito innate immune response is a major factor governing the interaction between vector and pathogen; consequently, it is a primary determinant of vector competence. Innate immunity in mosquitoes and other insects employs both cellular and humoral components in response to invading pathogens, but studies of the humoral aspects of innate immunity have dominated, with a primary emphasis on the identification of antimicrobial peptides (AMPs) and the signaling pathways involved in their production. Far less emphasis has been placed on hemocytes and cellular immune responses, but recent data clearly verify that cellular responses likely play a critical role in clearing microbial pathogens by phagocytosis, melanization, or by both processes in combination. There is a void in the data required to accurately define regulatory processes, hemocyte-produced effector molecules, and the role these cells and cell products play in innate immunity. It also is clear that AMPs are significant factors in innate immune responses against invading microbes, but it is not known how cellular and AMP-based humoral responses function in concert to clear microbial infections. We believe that a reductionist approach, that studies one gene, or a small set of genes, in relation to a phenotype, has limitations for determining the complex interrelationships that undoubtedly exist in mosquito innate immune responses. Therefore, in order to initate more holistic experimental approaches for studying innate immunity in mosquitoes, we developed a large expressed sequence tag (EST) data set from Aedes aegypti immune-activated hemocytes and complementary oligonucleotide-based microarrays as substrates to evaluate mosquito cellular, as well as humoral, immune responses. The general hypothesis for the research proposed herein is that mosquito innate immune responses are a highly complex network of processes that employ the distinct, but not mutually exclusive, responses of phagocytosis and melanization, in concert with AMPs, to clear bacteria infections from the hemolymph of infected mosquitoes. To test this hypothesis, we will use the mosquito Ae. aegypti and a number of bacteria species in (1) high-throughput molecular approaches to profile gene transcription and to identify distinct patterns underlying the anti-bacterial responses of phagocytosis and melanization, (2) utilize microarray analyses to assess transcription profile changes in relation to different bacterial species (that vary in their pathogenicity and ability to elicit a response) in order to better dissect mechanisms influencing cellular immunity, and (3) employ microarray analyses, synthetic peptides, RNAi methodologies, and phenotype evaluations to better define the role AMPs, and select unknown genes, play in innate immunity. All data resulting from microarray analyses, including experimental protocols used, will be made publicly available on the interactive website that contains our hemocyte EST data.