Layers of immunity: Deconstructing the

The host innate immune response relies on the cooperation of multiple defense modules. In insects and other arthropods, which have only innate immune mechanisms, four main immune-specific modules have been described in the defense against microbial invaders: the Toll pathway, the Imd pathway, the melanization response, and phagocytosis by plasmatocytes. Our present understanding of their relative importance remains fragmented as their contribution to host defense has never been simultaneously assessed across a large panel of pathogens. Here, we use newly described immune mutants in a controlled genetic background to systematically delete these four immune modules individually, in pairs, or even all four simultaneously. Surprisingly, flies simultaneously deficient in all four immune modules are viable (poor viability), homozygous fertile, and display no overt morphological defects, suggesting these immune mechanisms are not strictly required for organismal development. We assessed the contribution of each module individually and collectively against a diverse panel of viruses, fungi, and bacteria. We find these four modules largely function independently and additively in host defense. We could confirm previous findings on the importance of Imd and Toll, and their antimicrobial peptide and Bomanin (Bom) effectors, against relevant microbes. We also reveal a highly important role of melanization against viruses. Examining microbial load kinetics confirms how these modules contribute to resistance or tolerance against specific microbes. The set of immune-deficient lines provided here offers tools to better assess the role of these immune modules in host defense.