The melanization response is an important defense mechanism in arthropods. This reaction is mediated by phenoloxidases (POs), which are activated by complex extracellular serine protease (SP) cascades. Here, we investigate the role of SPs in the melanization response using compound mutants in D. melanogaster and discover phenotypes previously concealed in single-mutant analyses. We find that two SPs, Hayan and Sp7, activate the melanization response in different manners: Hayan is required for blackening wound sites, whereas Sp7 regulates an alternate melanization reaction responsible for the clearance of Staphylococcus aureus. We present evidence that Sp7 is regulated by SPs activating the Toll NF-κB pathway, namely ModSP and Grass. Additionally, we reveal a role for the combined action of Hayan and Psh in propagating Toll signaling downstream of pattern recognition receptors activating either Toll signaling or the melanization response.

Significance: Hosts sequester iron as a strategy to limit pathogen acquisition of this essential nutrient in a process termed nutritional immunity. Due to their in vitro antimicrobial activity, iron-binding proteins transferrins are suspected to play a role in iron sequestration. However, little is known about the in vivo role of transferrins. Here, we found thatDrosophila melanogasterexhibits infection-induced hypoferremia mediated by Transferrin 1. Due to excessive iron in hemolymph,Transferrin 1(Tsf1)-deficient flies are hypersusceptible to certain infections. Our study reveals that nutritional immunity is an important, previously unrecognized arm of immune defense inDrosophila. Using fly and bacterial genetics, we show that Tsf1 mediates nutritional immunity by sequestering iron from the pathogens in vivo on the whole-organism level.

The evolution of genetic mechanisms used to combat bacterial infections is critical for the survival of animals and plants, yet how these genes evolved to produce a robust defense system is poorly understood. Studies of the nematode Caenorhabditis elegans have uncovered a plethora of genetic regulators and effectors responsible for surviving pathogens. However, comparative studies utilizing other free-living nematodes and therefore providing an insight into the evolution of innate immunity have been lacking. Here, we take a systems biology approach and use whole genome microarrays to profile the transcriptional response of C. elegans and the necromenic nematode Pristionchus pacificus after exposure to the four different pathogens Serratia marcescens, Xenorhabdus nematophila, Staphylococcus aureus and Bacillus thuringiensis DB27. C. elegans is susceptible to all four pathogens whilst P. pacificus is only susceptible to S. marcescens and X. nematophila. We show an unexpected level of specificity in host responses to distinct pathogens within and across species, revealing an enormous complexity of effectors of innate immunity. Functional domains enriched in the transcriptomes on different pathogens are similar within a nematode species but different across them, suggesting differences in pathogen sensing and response networks. We find translation inhibition to be a potentially conserved response to gram-negative pathogens in both the nematodes. Further computational analysis indicates that both nematodes when fed on pathogens up-regulate genes known to be involved in other stress responses like heat shock, oxidative and osmotic stress, and genes regulated by DAF-16/FOXO and TGF-beta pathways. This study presents a platform for comparative systems analysis of two nematode model species, and a catalog of genes involved in the evolution of nematode immunity and identifies both pathogen specific and pan-pathogen responses. We discuss the potential effects of ecology on evolution of downstream effectors and upstream regulators on evolution of nematode innate immunity.

The fruit flyDrosophila melanogastercombats microbial infection by producing a battery of effector peptides that are secreted into the haemolymph. Technical difficulties prevented the investigation of these short effector genes until the recent advent of the CRISPR/CAS era. As a consequence, many putative immune effectors remain to be formally described, and exactly how each of these effectors contribute to survival is not well characterized. Here we describe a novelDrosophilaantifungal peptide gene that we nameBaramicin A. We show thatBaraAencodes a precursor protein cleaved into multiple peptides via furin cleavage sites.BaraAis strongly immune-induced in the fat body downstream of the Toll pathway, but also exhibits expression in other tissues. Importantly, we show that flies lackingBaraAare viable but susceptible to the entomopathogenic fungusBeauveria bassiana. Consistent withBaraAbeing directly antimicrobial, overexpression ofBaraApromotes resistance to fungi and the IM10-like peptides produced byBaraAsynergistically inhibit growth of fungi in vitro when combined with a membrane-disrupting antifungal. Surprisingly,BaraAmutant males but not females display an erect wing phenotype upon infection. Here, we characterize a new antifungal immune effector downstream of Toll signalling, and show it is a key contributor to theDrosophilaantimicrobial response.