Transcriptomics of phagocytosis and melanization in Aedes aegypti.

As is the case in vertebrates, successful mosquito immune responses depend on a complex and carefully orchestrated network of processes in order to defend the host from a foreign invader and to simultaneously maintain a homeostatic environment. Although commonalities exist between immune response mechanisms across phyla, invertebrates (e.g., mosquitoes) lack anticipatory, adaptive immune responses. However, a number of immune response elements, both cellular and humoral, exist to combat infection. Organisms that successfully invade the mosquito body cavity (hemocoel) potentially are subjected to a variety of mosquito defenses including: melanotic encapsulation, the production of antimicrobial peptides (AMPs) or reactive oxygen and nitrogen intermediates, or to engulfment by phagocytic cells. These arrays are used to temporally compare and contrast transcriptome profiles associated with these responses, as manifest in the cells (hemocytes) that circulate in the mosquito body cavity. Hemocytes have documented association with phagocytosis and melanization reactions in mosquitoes, and these responses, to a variety of Gram positive and Gram negative bacteria, have been characterized. Inoculated Escherichia coli elicit a strong phagocytic response, and Micrococcus luteus are rapidly melanized. For transcriptome profiling, inoculated moquitoes are compared to naïve (uninoculated) mosquitoes. Data obtained will begin to clarify the interactive role of hemocyte genes and gene products in orchestrating phagocytic, melanization, and AMP responses against invading bacteria. Keywords: time course Three-day old adult female mosquitoes were inoculated with ~500,000 bacteria from an overnight culture of either E. coli or M. luteus. For each sample, approximately 100 mosquitoes were inoculated, while an additional 100 from the same cohort were left as naive. Mosquito hemolymph was then perfused at 1, 8, or 24 hours post-inoculation. cDNA from treated and naive were labeled with Cy3 or Cy5 dye and hybridized on custom two-color chips. For each time point, and each inoculated species, at least four biological replicates from different cohorts were prepared. At least two technical replicates of each biological replicate were performed. to test dye-bias, each time point has at least one dye-swap technical replicate. A total of 51 chips were analyzed for this study.