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Coupling immunity and development is essential to ensure survival despite changing internal conditions in the organism. Drosophila metamorphosis represents a striking example of drastic and systemic physiological changes that need to be integrated with the innate immune system. However, nothing is known about the mechanisms that coordinate development and immune cell activity in the transition from larva to adult. Here, we reveal that regulation of macrophage-like cells (hemocytes) by the steroid hormone ecdysone is essential for an effective innate immune response over metamorphosis. Although it is generally accepted that steroid hormones impact immunity in mammals, their action on monocytes (e.g. macrophages and neutrophils) is still not well understood. Here in a simpler model system, we used an approach that allows in vivo, cell autonomous analysis of hormonal regulation of innate immune cells, by combining genetic manipulation with flow cytometry, high-resolution time-lapse imaging and tissue-specific transcriptomic analysis. We show that in response to ecdysone, hemocytes rapidly upregulate actin dynamics, motility and phagocytosis of apoptotic corpses, and acquire the ability to chemotax to damaged epithelia. Most importantly, individuals lacking ecdysone-activated hemocytes are defective in bacterial phagocytosis and are fatally susceptible to infection by bacteria ingested at larval stages, despite the normal systemic and local production of antimicrobial peptides. This decrease in survival is comparable to the one observed in pupae lacking immune cells altogether, indicating that ecdysone-regulation is essential for hemocyte immune functions and survival after infection. Microarray analysis of hemocytes revealed a large set of genes regulated at metamorphosis by EcR signaling, among which many are known to function in cell motility, cell shape or phagocytosis. This study demonstrates an important role for steroid hormone regulation of immunity in vivo in Drosophila, and paves the way for genetic dissection of the mechanisms at work behind steroid regulation of innate immune cells., Author Summary Hormones orchestrate physiological processes such as metabolism, immunity and development, and allow the body to respond systemically to external stresses and resources. Steroid hormones, such as sex hormones and corticosteroids, affect the function of the immune system, but how they do so is not well understood. The fruit fly Drosophila relies on the innate immune system to protect itself from infection by microbes. The high level of functional conservation of Drosophila immune cells, or hemocytes, with mammalian monocytes, and the powerful genetic tools makes the fly a useful model to uncover new aspects of innate immunity. Here, we have analyzed the regulation of hemocytes by the steroid hormone ecdysone at the moment when the juvenile larval form begins the process of metamorphosis, the drastic change of body plan that gives rise to an adult fly. We reveal that ecdysone rapidly activates hemocytes, increasing their ability to move, respond to wounds and engulf dead cells or invading bacteria. Hemocytes rendered insensitive to the hormone are not activated and as a result, the fly becomes susceptible to bacterial infections, compromising its survival. This study contributes to our understanding of steroid regulation of monocyte function, which has broad implications for human immunity.

Evolution of pathogen virulence is affected by the route of infection. Also, alternate infection routes trigger different physiological responses on hosts, impinging on host adaptation and on its interaction with pathogens. Yet, how route of infection may shape adaptation to pathogens has not received much attention at the experimental level. We addressed this question through the experimental evolution of an outbred Drosophila melanogaster population infected by two different routes (oral and systemic) with Pseudomonas entomophila. The two selection regimes led to markedly different evolutionary trajectories. Adaptation to infection through one route did not protect from infection through the alternate route, indicating distinct genetic bases. Finally, relatively to the control population, evolved flies were not more resistant to bacteria other than Pseudomonas and showed higher susceptibility to viral infections. These specificities and trade-offs may contribute to the maintenance of genetic variation for resistance in natural populations. Our data shows that the infection route affects host adaptation and thus, must be considered in studies of host-pathogen interaction., Author Summary Pathogens enter their hosts through several routes, the most common being ingestion (oral infection) and breaches in the cuticle (systemic infection). Several studies have shown that these infection routes strongly affect the evolution of pathogen virulence, though little attention has been given to the role of host evolution in this process. Here, we study the effect of infection route on the evolution of host defenses, using Drosophila melanogaster and its natural pathogen Pseudomonas entomophila. Profiting from the power of experimental evolution, in which the evolution of populations is followed in real time, we show that survival of D. melanogaster to an oral infection increases within the first 3 generations of selection, whereas the response to systemic infection is slower. Furthermore, we show that the evolved response is specific to the route of infection and to pathogen. Indeed, flies that resist bacteria through ingestion are not protected from systemic infection with the same bacteria species, and vice versa. Also, evolution of resistance to one pathogen does not extend to infections with bacteria of different genera via the same infection route. This degree of specificity calls for more attention onto pathogen infection routes in studies of host-parasite interactions.