The prophenoloxidase-activating cascade is a key component of arthropod immunity. Drosophila prophenoloxidase is stored in crystal cells, a specialized class of blood cells from which it is released through cell rupture. Within minutes after bleeding, prophenoloxidase is activated leading to visible melanization of the clot matrix. Using crystal cell rupture and melanization as readouts to screen mutants in signal transduction pathways, we show that prophenoloxidase release requires Jun N-terminal kinase, small Rho GTPases and Eiger, the Drosophila homolog of tumor necrosis factor. We also provide evidence that in addition to microbial products, endogenous signals from dying hemocytes contribute to triggering and/or assembly of the prophenoloxidase-activating cascade, and that this process can be inhibited in vitro and in vivo using the viral apoptotic inhibitor p35. Our results provide a more comprehensive view of immune signal transduction pathways, with implications for immune reactions where cell death is used as a terminal mode of cell activation.

Clotting systems are required in almost all animals to prevent loss of body fluids after injury. Here, we show that despite the risks associated with its systemic activation, clotting is a hitherto little appreciated branch of the immune system. We compared clotting of human blood and insect hemolymph to study the best-conserved component of clotting systems, namely the Drosophila enzyme transglutaminase and its vertebrate homologue Factor XIIIa. Using labelled artificial substrates we observe that transglutaminase activity from both Drosophila hemolymph and human blood accumulates on microbial surfaces, leading to their sequestration into the clot. Using both a human and a natural insect pathogen we provide functional proof for an immune function for transglutaminase (TG). Drosophila larvae with reduced TG levels show increased mortality after septic injury. The same larvae are also more susceptible to a natural infection involving entomopathogenic nematodes and their symbiotic bacteria while neither phagocytosis, phenoloxidase or—as previously shown—the Toll or imd pathway contribute to immunity. These results firmly establish the hemolymph/blood clot as an important effector of early innate immunity, which helps to prevent septic infections. These findings will help to guide further strategies to reduce the damaging effects of clotting and enhance its beneficial contribution to immune reactions. One of the main functions of immune systems is to prevent the dissemination of microbes and the resulting sepsis. Blood clotting during sepsis has until now been primarily regarded as harmful, leading to the formation of widespread clots in blood vessels and as a result to organ failure. Here we show that clotting also has a protective function to limit and prevent infections. This is achieved by capturing bacteria in the clot. Our infection studies were performed in the insect model Drosophila melanogaster where, due to the presence of an open circulatory system, the negative effects of clotting are less pronounced. We show that clotting of hemolymph—the insect blood equivalent—is essential in Drosophila to prevent septic death arising from injection of bacteria or infection with a natural pathogen. We also show that both Drosophila transglutaminase and its human homologue clotting factor XIII are key enzymes for sequestration of bacteria in the clot matrix, indicating the conserved nature of the clot's function in immunity. Our data are expected to lead to a much stronger appreciation of the role of blood clotting in innate immunity, and will guide future therapies which target this process.

Phylogenetically conserved serine protease cascades play an important role in invertebrate and vertebrate immunity. The mammalian coagulation system can be traced back some 400 million years and shares homology with ancestral serine proteinase cascades that are involved in, for example, Toll receptor signaling in insects and release of antimicrobial peptides during hemolymph clotting. In the present study, we show that the induction of coagulation by bacteria leads to immobilization and killing of Streptococcus pyogenes bacteria inside the clot. The entrapment is mediated via cross-linking of bacteria to fibrin fibers by the action of coagulation factor XIII (fXIII), an evolutionarily conserved transglutaminase. In a streptococcal skin infection model, fXIII^−/− mice developed severe signs of pathologic inflammation at the local site of infection, and fXIII treatment of wild-type animals dampened bacterial dissemination during early infection. Bacterial killing and cross-linking to fibrin networks was also detected in tissue biopsies from patients with streptococcal necrotizing fasciitis, supporting the concept that coagulation is part of the early innate immune system.

Some organisms can adapt to seasonal and other environmental challenges by entering a state of dormancy, diapause. Thus, insects exposed to decreased temperature and short photoperiod enter a state of arrested development, lowered metabolism, and increased stress resistance. Drosophila melanogaster females can enter a shallow reproductive diapause in the adult stage, which drastically reduces organismal senescence, but little is known about the physiology and endocrinology associated with this dormancy, and the genes involved in its regulation. We induced diapause in D. melanogaster and monitored effects over 12 weeks on dynamics of ovary development, carbohydrate and lipid metabolism, as well as expression of genes involved in endocrine signaling, metabolism and innate immunity. During diapause food intake diminishes drastically, but circulating and stored carbohydrates and lipids are elevated. Gene transcripts of glucagon- and insulin-like peptides increase, and expression of several target genes of these peptides also change. Four key genes in innate immunity can be induced by infection in diapausing flies, and two of these, drosomycin and cecropin A1, are upregulated by diapause independently of infection. Diapausing flies display very low mortality, extended lifespan and decreased aging of the intestinal epithelium. Many phenotypes induced by diapause are reversed after one week of recovery from diapause conditions. Furthermore, mutant flies lacking specific insulin-like peptides (dilp5 and dilp2-3) display increased diapause incidence. Our study provides a first comprehensive characterization of reproductive diapause in D. melanogaster, and evidence that glucagon- and insulin-like signaling are among the key regulators of the altered physiology during this dormancy.