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WIREs Dev Biol
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Methods for the study of innate immunity in Drosophila melanogaster

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From flies to humans, many components of the innate immune system have been conserved during metazoan evolution. This foundational observation has allowed us to develop Drosophila melanogaster, the fruit fly, into a powerful model to study innate immunity in animals. Thanks to an ever‐growing arsenal of genetic tools, an easily manipulated genome, and its winning disposition, Drosophila is now employed to study not only basic molecular mechanisms of pathogen recognition and immune signaling, but also the nature of physiological responses activated in the host by microbial challenge and how dysregulation of these processes contributes to disease. Here, we present a collection of methods and protocols to challenge the fly with an assortment of microbes, both systemically and orally, and assess its humoral, cellular, and epithelial response to infection. Our review covers techniques for measuring the reaction to microbial infection both qualitatively and quantitatively. Specifically, we describe survival, bacterial load, BLUD (a measure of disease tolerance), phagocytosis, melanization, clotting, and ROS production assays, as well as efficient protocols to collect hemolymph and measure immune gene expression. We also offer an updated catalog of online resources and a collection of popular reporter lines and mutants to facilitate research efforts. This article is categorized under: Technologies > Analysis of Cell, Tissue, and Animal Phenotypes
The response to infection in Drosophila melanogaster. Schematic overview of Drosophila host defense. Detection of an array of elicitors triggers a coordinated and synergistic activation of defense modules in the fly. * Denotes reporter lines associated with select pathways. ● Indicates reporter genes linked to individual pathways. Detailed information on these reporter lines and reporter genes can be found in Tables and , respectively
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Tracking phagocytosis in vivo. Fluorescent images of the abdomen following injection with pHrodo red E. coli, which allows for visualization of phagocytosis in vivo. pHrodo red bacteria are nonfluorescent outside the cell, but fluoresce brightly red once inside phagosomes. Abdomen of wildtype fly and abdomen of phagocytosis‐deficient crq mutant fly
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Qualitative and quantitative methods of pathogen detection. (a) Schematic representation of a model for bacterial growth within the host during the course of a chronic infection. In the early phase of infection, which precedes effective control by the immune system, bacteria grow exponentially inside the host. This is followed by the second stage of infection, called resolution, in which some of the hosts start to control bacterial proliferation lowering their total CFU (colony forming units) load. Hosts that fail to control their pathogen load early on entering the terminal phase of infection, where bacteria continue to divide until reaching a load that cannot be sustained by the host. Upon reaching this load, termed the bacterial load upon death or BLUD, the host dies. Hosts that survive the infection by controlling their bacterial burden enter the chronic phase of infection, where they sustain a persistent pathogen load called the set‐point bacterial load (SPBL). To measure bacterial load, live flies can be sampled at points of interest during both the early and resolution phases of infection. To measure the BLUD, dead flies are collected within 15 min of their death. To quantify the SPBL, live flies should be sampled during the chronic phase of infection (approximately 5 days after infection is a good starting point for bacteria such as P. rettgeri and E. faecalis). (b) To quantify bacteria, individual flies are placed in single Eppendorf tubes containing 500 μL of sterile PBS buffer and a metal bead. The sample is then homogenized. (c) Following homogenization, samples are plated using the appropriate agar media. We advise that at least two dilutions of each sample are plated the first time an experiment is conducted so as to ensure that individual colonies are discernible and therefore countable. (d) Drosophila larvae orally infected with Ecc15‐GFP, which is visible in the gut of the larvae. (e) Gut of an adult fly orally infected with Ecc15‐GFP
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Tracking midgut renewal following enteric infection. Fluorescent reporter line esgF/O, which strongly labels intestinal stem cells, enteroblasts, and newly synthesized enterocytes, can be used to detect midgut renewal following oral infection with a pathogen. (a) Unchallenged adult midgut. (b) Adult midgut after enteric infection with Ecc15
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Drosophila infection techniques. (a) Infection station setup. (b) Anesthetized fly pricked in the thorax with a stainless steel needle. (c) Fly injected in the abdomen with a defined volume using a microinjector. (d) Flies coated in fungal spores during natural infection. Two methods are available for oral infection. (e) In the first, previously starved flies are orally infected using a precut Whatman Filter Paper disk soaked in a bacterial solution. The paper disk sits atop food in a fly vial. This placement ensures that the flies receive the inoculum as well as nutrients, which are absorbed through the filter. (f) The second protocol places flies in a vial devoid of food, where they feed on a small piece of a Tork napkin that has been soaked in a bacterial solution. The availability of a nutrient source beyond the bacterial solution distinguishes the two methods
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