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.

Heterorhabditis bacteriophora is an entomopathogenic nematode (EPN) which infects its host by accessing the hemolymph where it releases endosymbiotic bacteria of the species Photorhabdus luminescens. We performed a genome-wide transcriptional analysis of the Drosophila response to EPN infection at the time point at which the nematodes reached the hemolymph either via the cuticle or the gut and the bacteria had started to multiply. Many of the most strongly induced genes have been implicated in immune responses in other infection models. Mapping of the complete set of differentially regulated genes showed the hallmarks of a wound response, but also identified a large fraction of EPN-specific transcripts. Several genes identified by transcriptome profiling or their homologues play protective roles during nematode infections. Genes that positively contribute to controlling nematobacterial infections encode: a homolog of thioester-containing complement protein 3, a basement membrane component (glutactin), a recognition protein (GNBP-like 3) and possibly several small peptides. Of note is that several of these genes have not previously been implicated in immune responses.

Background: The basic function of the immune system is to protect an organism against infection in order to minimize the fitness costs of being infected. According to life-history theory, energy resources are in a trade-off between the costly demands of immunity and other physiological demands. Concerning fish, both physiology and immunity are influenced by seasonal changes (i.e. temporal variation) associated to the changes of abiotic factors (such as primarily water temperature) and interactions with pathogens and parasites. In this study, we investigated the potential associations between the physiology and immunocompetence of common carp (Cyprinus carpio) collected during five different periods of a given year. Our sampling included the periods with temporal variability and thus, it presented a different level in exposure to parasites. We analyzed which of two factors, seasonality or parasitism, had the strongest impact on changes in fish physiology and immunity. Results: We found that seasonal changes play a key role in affecting the analyzed measurements of physiology, immunity and parasitism. The correlation analysis revealed the relationships between the measures of overall host physiology, immunity and parasite load when temporal variability effect was removed. When analyzing separately parasite groups with different life-strategies, we found that fish with a worse condition status were infected more by monogeneans, representing the most abundant parasite group. The high infection by cestodes seems to activate the phagocytes. A weak relationship was found between spleen size and abundance of trematodes when taking into account seasonal changes. Conclusions: Even if no direct trade-off between the measures of host immunity and physiology was confirmed when taking into account the seasonality, it seems that seasonal variability affects host immunity and physiology through energy allocation in a trade-off between life important functions, especially reproduction and fish condition. Host immunity measures were not found to be in a trade-off with the investigated physiological traits or functions, but we confirmed the immunosuppressive role of 11-ketotestosterone on fish immunity measured by complement activity. We suggest that the different parasite life-strategies influence different aspects of host physiology and activate the different immunity pathways.