The Larval Clot and Immune Defense. Clara I. Bajzek, Mitchell S. Dushay. BCHS, Illinois Institute of Technology, Chicago, Il.

   Since the identification of larval clotting factors in Drosophila, experiments have been performed to study how they interact to form the clot and to understand the clot's physiological and immune functions. Loss of the major clotting factor Hemolectin abolishes coagulation in vitro, but hml mutant larvae show surprisingly small decreases in wound survival. This indicated the presence of other, still-poorly understood hemostatic mechanisms, and the limited value of wound survival as an in vivo clotting assay. We developed a method using microcapillary tubes to measure how much injured larvae bleed. The capillary assay revealed a greater difference between hml and control larvae than shown by wound survival. To further reduce coagulation, we tested fon; hml double mutants. To our surprise, these larvae survived wounding as well as hml alone. Loss of fon altered the clot's physical properties, but apparently did not reduce coagulation. Fondue is a major substrate for transglutaminase - the only clotting factor shared by Drosophila and vertebrates (factor XIIIA). Transglutaminase RNAi knockdown reduced coagulation by in vitro assay and these larvae were more susceptible to infection. Yet hml mutant larvae were not consistently more prone to infection, making it difficult to evaluate the importance of the clot in immune defense. These seemingly conflicting results were obtained using different bacterial challenges and different protocols. We have tested feeding and wandering stage third instar larvae bearing combinations of mutations in hml, fon, tiggrin, Eig71Ee, and transglutaminase RNAi knockdown by in vitro and in vivo clot assays and by needle puncture infection to learn more about how Hemolectin and Transglutaminase work together, the nature of the clot, and its involvement in immune defense.