Spatio-temporal characterization of the insulin signaling cascade and its role in regulating hemocyte proliferation in Aedes aegypti
Castillo, Julio Cesar
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Hemocytes are central for cell based immunity. In this study, I compared an improved technique (this study) to collect hemocytes from mosquitoes to other welle stablished methods. Collection method greatly affected the number of hemocytes and contaminants obtained from adult females of each species. Using a collection method called high injection/recovery I was able to show that hemolymph from An. gambiae and Ae. aegypti adult females contains three hemocyte types (granulocytes, oenocytoids and prohemocytes) that were distinguished from one another by a combination of morphological and functional markers. Granulocytes were the most abundant cell type in both species while oenocytoids and prohemocytes comprised less than 10% of the total hemocyte population. The number of hemocytes recovered from sugar fed females declined with age but blood feeding transiently increased hemocyte abundance. In order to understand the nature of the increase in blood cells after a blood meal, the role of insulin signaling as key regulator of cell proliferation was investigated. Several brain-specific ILPs (-3, -4, -7, and -8) were found to be expressed in hemocytes, and their expression pattern differ between blood fed and non-blood fed female mosquitoes. Experiments showed that decapitated females exhibited no increase in hemocyte abundance. ILP-3 injected into blood fed/decapitated females rescued the observed cell increase phenotype, and hemocyte increase was restored. BrdU labeling of hemocytes showed that blood fed females had higher numbers of BrdU positive cells when compared to non blood fed controls, indicating that blood feeding stimulates cell proliferation. RNAi knock down using dsRNA targeting the mosquito insulin receptor (MIR) inhibited hemocyte increases. Additionally, the total hemocyte number in dsRNA-MIR treated blood fed and non-blood fed females was considerably lower, suggesting that ILPs may also serve as a survival signal. Lastly I found that the increase in hemocyte numbers observed in blood fed animals confered resistance to bacterial infection.