Healthy Flies Research Articles

Notch signaling promotes differentiation, cell death and autophagy in Drosophila hematopoietic system

Notch signaling is a highly conserved pathway between mammals and Drosophila and plays a key role in various biological processes. Drosophila has emerged as a powerful model for studying hematopoiesis and leukemia. In exception to crystal cells, the strength of Notch signaling in Drosophila lymph gland cortical zone (CZ)/intermediate zone (IZ) cells is weak. However, the influence of Notch activation in the lymph gland CZ/IZ cells and circulating hemocytes on hematopoietic homeostasis maintenance is unclear. Here, we showed that Notch activation in lymph gland CZ/IZ cells induced overdifferentiation of progenitors. Moreover, Notch activation promoted lamellocyte generation via NFκB/Toll signaling activation and increased reactive oxygen species (ROS). In addition, we found that Notch activation in lymph gland CZ/IZ cells and circulating hemocytes caused caspase-independent and nonautophagic cell death. However, crystal cell autophagy was activated by upregulation of the expression of the target gene of the Hippo/Yki pathway Diap1. Moreover, we showed that Notch activation could alleviate cytokine storms and improve the survival of Rasv12 leukemia model flies. Our study revealed the various mechanisms of hematopoietic dysregulation induced by Notch activation in healthy flies and the therapeutic effect of Notch activation on leukemia model flies.

Gut cancer increases the risk of Drosophila being preyed upon by hunting spiders

Predators are thought to prey on individuals that are in poor physical condition, although the evidence supporting this is ambiguous. We tested whether sick individuals were more predated using Drosophila melanogaster flies as manipulable prey. We asked whether hunting spiders, trapped from the wild, would selectively prey upon flies with compromised health (i.e. chronically infected or cancerous) versus healthy flies, under laboratory conditions. Flies chronically infected with the bacterium Providencia rettgeri , a natural Drosophila pathogen, were not selectively preyed upon by jumping spiders. We strengthened and confirmed our finding with another hunting spider species, small wolf spiders. This result supports the hypothesis that chronic infection is associated with reduced symptoms notably to avoid the potentially deadly consequences for pathogens of host predation. We then induced colon cancer in some of the flies and asked whether the presence of cancer led to selective predation; there is little empirical evidence for this, even in vertebrates. As the cancer developed, the incidence of predation by jumping spiders on the afflicted flies increased. We conclude that disease can have different lethal consequences through predation, even in invertebrate species, and that cancer is a factor in selective predation. Our results may explain why early tumours, but not metastasized cancers, are commonly detected in organisms in the wild, as cancer-bearing individuals are rapidly eliminated due to the strong selective pressure against them. • Chronic infection of Drosophila did not significantly affect the risk of predation. • Predation may play a role in selection of symptoms of chronic infections. • Hyperplasia in Drosophila gut did not change predation risk by jumping spiders. • Advanced cancer in Drosophila gut increased predation risk by jumping spiders. • Selective predation may explain why cancer is rarely detected in wild populations.

Bacterial recognition by PGRP-SA and downstream signalling by Toll/DIF sustain commensal gut bacteria in Drosophila.

The gut sets the immune and metabolic parameters for the survival of commensal bacteria. We report that in Drosophila, deficiency in bacterial recognition upstream of Toll/NF-κB signalling resulted in reduced density and diversity of gut bacteria. Translational regulation factor 4E-BP, a transcriptional target of Toll/NF-κB, mediated this host-bacteriome interaction. In healthy flies, Toll activated 4E-BP, which enabled fat catabolism, which resulted in sustaining of the bacteriome. The presence of gut bacteria kept Toll signalling activity thus ensuring the feedback loop of their own preservation. When Toll activity was absent, TOR-mediated suppression of 4E-BP made fat resources inaccessible and this correlated with loss of intestinal bacterial density. This could be overcome by genetic or pharmacological inhibition of TOR, which restored bacterial density. Our results give insights into how an animal integrates immune sensing and metabolism to maintain indigenous bacteria in a healthy gut.

A mitochondrial membrane-bridging machinery mediates signal transduction of intramitochondrial oxidation.

Mitochondria are the main site for generating reactive oxygen species, which are key players in diverse biological processes. However, the molecular pathways of redox signal transduction from the matrix to the cytosol are poorly defined. Here we report an inside-out redox signal of mitochondria. Cysteine oxidation of MIC60, an inner mitochondrial membrane protein, triggers the formation of disulfide bonds and the physical association of MIC60 with Miro, an outer mitochondrial membrane protein. The oxidative structural change of this membrane-crossing complex ultimately elicits cellular responses that delay mitophagy, impair cellular respiration and cause oxidative stress. Blocking the MIC60-Miro interaction or reducing either protein, genetically or pharmacologically, extends lifespan and health-span of healthy fruit flies, and benefits multiple models of Parkinson's disease and Friedreich's ataxia. Our discovery provides a molecular basis for common treatment strategies against oxidative stress.

Antimicrobial Effect of Roselle (Hibiscus sabdariffa L.) Water Fraction against Pseudomonas aeruginosa using Drosophila Infection Model

Pseudomonas aeruginosa is one of the most common pathogenic bacteria that cause nosocomial infection. Unfortunately, the irrational use of antibiotics has created a surge in P. aeruginosa resistance nowadays. To overcome this situation, new antibacterial compounds are urgently needed. One of the potential sources to obtain such antibacterial compounds is roselle calyx. This research was carried out using two experimental approaches, survival assay and gene expression analysis, to examine the in vivo antibacterial effect of water fraction of roselle calyx (WFR) against Pseudomonas aeruginosa in Drosophila model of infection. Survival assay was used to demonstrate the impact of treatment on the lifespan of the infected host. The measurement of immune-related Dpt mRNA levels by reverse-transcriptase quantitative PCR (RT-qPCR) was used to assess whether immunostimulation is involved in the antibacterial protection of WFR against P. aeruginosa. The result demonstrated that WFR at concentrations of 0.8% and 2% were able to enhance P. aeruginosa-infected flies' survival. Furthermore, gene expression analysis showed the insignificant difference between WFR-treated flies and healthy control flies at all tested concentrations, implying the non-involvement of Imd-Dpt-mediated pathway immunity in the antipseudomonal protection of WFR. Taken together, our data suggested the in vivo antibacterial activity of WFR against P. aeruginosa in the fruit fly model of infection.

The Effect of the Musca domestica Salivary Gland Hypertrophy Virus on Food Consumption in Its Adult Host, the Common House Fly (Diptera: Muscidae).

The Musca domestica salivary gland hypertrophy virus (MdSGHV) substantially enlarges the house fly's salivary glands and prevents or delays ovarian development in its adult host, but the effect that MdSGHV has on the house fly's food consumption is currently unknown. Using house flies from a laboratory-reared colony, we evaluated the effect of MdSGHV infection on food consumption over a 7-d period. Both treatment (virus-infected) and control (saline-injected) flies were provided with a choice of 8% sucrose solution and 4% powdered milk solution to determine food preferences. Quantities of each solution consumed were measured every 24 h for each fly to measure food consumptions. Infected house flies were shown to consume less overall of both solutions than house flies injected with saline. The largest consumption discrepancy was seen between female house flies. Healthy female flies with developing ovaries continued to consume a sugar and protein diet, whereas infected female flies fed predominantly on a sugar diet. Additionally, infected male and female flies consumed significantly lower quantities of protein and sucrose than control flies. This suggests that MdSGHV has a negative consumption effect (e.g., hunger, starvation) on its host. Thus, differences in food consumption of infected and control flies probably represent differences in the nutritional requirements of flies resulting from viral infection.

Examining effects of E. muscae on olfactory function in D. melanogaster

Many parasites manipulate the behavior of their hosts to induce specific behavior for the benefit of the parasite (i.e., to increase its reproduction and transmission). The complex ability to take over a host’s central nervous system has been studied extensively in ants (Camponotus castaneus) infected by the fungus Ophiocordyceps unilateralis s.l., but not as much in fruit flies (Drosophila melanogaster) infected by the fungus Entomophthora muscae. One behavioral change seen in ants infected by O. unilateralis s.l. is a reduced response to olfactory stimuli; however, this has yet to be tested in infected fruit flies. In this study, I tested olfactory response in flies infected with E. muscae, healthy flies (positive control), and flies without odorant receptors (negative control) via a preference experiment using a saturated sucrose solution and distilled water. The results supported the hypothesis that fruit flies do have a reduced perception of olfactory stimuli when exposed to E. muscae, as they perform more like mutant flies without odorant receptors than healthy flies with a functioning olfactory system (p < 0.05). Although, it is not fully understood how E. muscae enters and takes control of the fruit flies’ nervous system, knowing that E. muscae reduces their olfaction gives us more insight into the functionality of this parasite overall. Further research that allows us to understand how a nervous system is overtaken could potentially provide insight into the vulnerable parts of the nervous system and thus deepen our understanding of neurology as a whole.

A conserved role for sleep in supporting Spatial Learning in Drosophila.

Sleep loss and aging impair hippocampus-dependent Spatial Learning in mammalian systems. Here we use the fly Drosophila melanogaster to investigate the relationship between sleep and Spatial Learning in healthy and impaired flies. The Spatial Learning assay is modeled after the Morris Water Maze. The assay uses a "thermal maze" consisting of a 5 × 5 grid of Peltier plates maintained at 36-37°C and a visual panorama. The first trial begins when a single tile that is associated with a specific visual cue is cooled to 25°C. For subsequent trials, the cold tile is heated, the visual panorama is rotated and the flies must find the new cold tile by remembering its association with the visual cue. Significant learning was observed with two different wild-type strains-Cs and 2U, validating our design. Sleep deprivation prior to training impaired Spatial Learning. Learning was also impaired in the classic learning mutant rutabaga (rut); enhancing sleep restored learning to rut mutants. Further, we found that flies exhibited a dramatic age-dependent cognitive decline in Spatial Learning starting at 20-24 days of age. These impairments could be reversed by enhancing sleep. Finally, we find that Spatial Learning requires dopaminergic signaling and that enhancing dopaminergic signaling in aged flies restored learning. Our results are consistent with the impairments seen in rodents and humans. These results thus demonstrate a critical conserved role for sleep in supporting Spatial Learning, and suggest potential avenues for therapeutic intervention during aging.

Comparing the Effects of Different Natural Products on Reducing Tumor Growth in a Drosophila Model

Cancer is one of the main causes of death in the present day and due to side-effects on current chemotherapies there is a need for testing natural products in cancer studies. In this work, we compared the effects of common natural products, including sesame, cinnamon, garlic, moringa and turmeric. The tumor model was a comparison of “rough-eye” versus “smooth-eye” studied in Drosophila eye, which provides easy detection of the phenotypes via simple microscopy. Our data showed that these natural products cannot be used to reduce tumors once it has completely formed. However, our data confirmed that these natural products can be used to reduce cancer cell growth when treated early. Moringa and sesame helped reduce tumor size when used in right concentrations. Our study also revealed a 15% moringa solution is the best candidate, of the ones tested, to prevent tumors. Flies treated with moringa showed longer eye lengths and widths compared to the control sample and close to healthy wild flies. For the flies treated with moringa, the number of flies with smooth-eye and uniform ommatidia increased with natural product concentration until 15% and decreased at 20%. Garlic, turmeric and cinnamon products did not show much efficacy on the tumor reduction. Though turmeric treatment did not prevent cancer, it helped increase the lifespan compared to control samples and other natural products.

Fungal artillery of zombie flies: infectious spore dispersal using a soft water cannon.

Dead sporulating female fly cadavers infected by the house fly-pathogenic fungus Entomophthora muscae are attractive to healthy male flies, which by their physical inspection may mechanically trigger spore release and by their movement create whirlwind airflows that covers them in infectious conidia. The fungal artillery of E. muscae protrudes outward from the fly cadaver, and consists of a plethora of micrometric stalks that each uses a liquid-based turgor pressure build-up to eject a jet of protoplasm and the initially attached spore. The biophysical processes that regulate the release and range of spores, however, are unknown. To study the physics of ejection, we design a biomimetic 'soft cannon' that consists of a millimetric elastomeric barrel filled with fluid and plugged with a projectile. We precisely control the maximum pressure leading up to the ejection, and study the cannon efficiency as a function of its geometry and wall elasticity. In particular, we predict that ejection velocity decreases with spore size. The calculated flight trajectories under aerodynamic drag predict that the minimum spore size required to traverse a quiescent layer of a few millimetres around the fly cadaver is approximately 10 µm. This corroborates with the natural size of E. muscae conidia (approx. 27 µm) being large enough to traverse the boundary layer but small enough (less than 40 µm) to be lifted by air currents. Based on this understanding, we show how the fungal spores are able to reach a new host.

Effect of Metarhizium guizhouense infection on mating competition and mate choice of Bactrocera latifrons (Diptera: Tephritidae)

Metarhizium guizhouense PSUM02 treated males of Bactrocera latifrons were investigated for the mating competition among males and mating choice by female flies to develop an auto-dissemination for the control of B. latifrons. In the present study, on day 1–4 of experiment, M. guizhouense–treated male flies were equally competitive with the normal male flies as we did not observe any differences in mating by treated and normal male flies of B. latifrons. Further, mating competitiveness were found low in treated adult male B. latifrons than normal male B. latifrons from 5th days of treatment until death. Kaplan-Meier survival analysis of treated male flies gave average survival times (AST) of 4.3 ± 0.1 days, while the healthy female and male flies in the same cage showed AST of 9.3 ± 0.3 and 8.3 ± 0.4 days, respectively. The AST of untreated flies in control experiment ranged from 14.2–14.5 days. In mating preference experiment, M. guizhouense–treated male flies were chosen by virgin female than gravid female flies for mating. The treated male flies caused mortality in both virgin and gravid female flies in the same cage with AST of 4.4 ± 0.1, 5.6 ± 0.1 and 7.4 ± 0.2 days, respectively, while untreated flies showed AST ranged from 13.9–14.3 days in control. The treated male flies could transmit the fungus infection to both untreated female and male flies as well as in virgin and gravid female flies by mating and contact. Our experiments showed the potentiality of M. guizhouense PSUM02 in management of B. latifrons by auto-dissemination with treated male flies, which transmit the fungus to a healthy population to reduce insect pest infestations.

Pathogenic bacteria enhance dispersal through alteration of Drosophila social communication

Pathogens and parasites can manipulate their hosts to optimize their own fitness. For instance, bacterial pathogens have been shown to affect their host plants’ volatile and non-volatile metabolites, which results in increased attraction of insect vectors to the plant, and, hence, to increased pathogen dispersal. Behavioral manipulation by parasites has also been shown for mice, snails and zebrafish as well as for insects. Here we show that infection by pathogenic bacteria alters the social communication system of Drosophila melanogaster. More specifically, infected flies and their frass emit dramatically increased amounts of fly odors, including the aggregation pheromones methyl laurate, methyl myristate, and methyl palmitate, attracting healthy flies, which in turn become infected and further enhance pathogen dispersal. Thus, olfactory cues for attraction and aggregation are vulnerable to pathogenic manipulation, and we show that the alteration of social pheromones can be beneficial to the microbe while detrimental to the insect host.

Interaction Between Familial Transmission and a Constitutively Active Immune System Shapes Gut Microbiota in Drosophila melanogaster.

Resident gut bacteria are constantly influencing the immune system, yet the role of the immune system in shaping microbiota composition during an organism’s life span has remained unclear. Experiments in mice have been inconclusive due to differences in husbandry schemes that led to conflicting results. We used Drosophila as a genetically tractable system with a simpler gut bacterial population structure streamlined genetic backgrounds and established cross schemes to address this issue. We found that, depending on their genetic background, young flies had microbiota of different diversities that converged with age to the same Acetobacteraceae-dominated pattern in healthy flies. This pattern was accelerated in immune-compromised flies with higher bacterial load and gut cell death. Nevertheless, immune-compromised flies resembled their genetic background, indicating that familial transmission was the main force regulating gut microbiota. In contrast, flies with a constitutively active immune system had microbiota readily distinguishable from their genetic background with the introduction and establishment of previously undetectable bacterial families. This indicated the influence of immunity over familial transmission. Moreover, hyperactive immunity and increased enterocyte death resulted in the highest bacterial load observed starting from early adulthood. Cohousing experiments showed that the microenvironment also played an important role in the structure of the microbiota where flies with constitutive immunity defined the gut microbiota of their cohabitants. Our data show that, in Drosophila, constitutively active immunity shapes the structure and density of gut microbiota.

NF-κB Immunity in the Brain Determines Fly Lifespan in Healthy Aging and Age-Related Neurodegeneration

SummaryDuring aging, innate immunity progresses to a chronically active state. However, what distinguishes those that “age well” from those developing age-related neurological conditions is unclear. We used Drosophila to explore the cost of immunity in the aging brain. We show that mutations in intracellular negative regulators of the IMD/NF-κB pathway predisposed flies to toxic levels of antimicrobial peptides, resulting in early locomotor defects, extensive neurodegeneration, and reduced lifespan. These phenotypes were rescued when immunity was suppressed in glia. In healthy flies, suppressing immunity in glial cells resulted in increased adipokinetic hormonal signaling with high nutrient levels in later life and an extension of active lifespan. Thus, when levels of IMD/NF-κB deviate from normal, two mechanisms are at play: lower levels derepress an immune-endocrine axis, which mobilizes nutrients, leading to lifespan extension, whereas higher levels increase antimicrobial peptides, causing neurodegeneration. Immunity in the fly brain is therefore a key lifespan determinant.

Early-onset sleep defects in Drosophila models of Huntington's disease reflect alterations of PKA/CREB signaling.

Huntington's disease (HD) is a progressive neurological disorder whose non-motor symptoms include sleep disturbances. Whether sleep and activity abnormalities are primary molecular disruptions of mutant Huntingtin (mutHtt) expression or result from neurodegeneration is unclear. Here, we report Drosophila models of HD exhibit sleep and activity disruptions very early in adulthood, as soon as sleep patterns have developed. Pan-neuronal expression of full-length or N-terminally truncated mutHtt recapitulates sleep phenotypes of HD patients: impaired sleep initiation, fragmented and diminished sleep, and nighttime hyperactivity. Sleep deprivation of HD model flies results in exacerbated sleep deficits, indicating that homeostatic regulation of sleep is impaired. Elevated PKA/CREB activity in healthy flies produces patterns of sleep and activity similar to those in our HD models. We were curious whether aberrations in PKA/CREB signaling were responsible for our early-onset sleep/activity phenotypes. Decreasing signaling through the cAMP/PKA pathway suppresses mutHtt-induced developmental lethality. Genetically reducing PKA abolishes sleep/activity deficits in HD model flies, restores the homeostatic response and extends median lifespan. In vivo reporters, however, show dCREB2 activity is unchanged, or decreased when sleep/activity patterns are abnormal, suggesting dissociation of PKA and dCREB2 occurs early in pathogenesis. Collectively, our data suggest that sleep defects may reflect a primary pathological process in HD, and that measurements of sleep and cAMP/PKA could be prodromal indicators of disease, and serve as therapeutic targets for intervention.

In vivo CaspaseTracker biosensor system for detecting anastasis and non-apoptotic caspase activity.

The discovery that mammalian cells can survive late-stage apoptosis challenges the general assumption that active caspases are markers of impending death. However, tools have not been available to track healthy cells that have experienced caspase activity at any time in the past. Therefore, to determine if cells in whole animals can undergo reversal of apoptosis, known as anastasis, we developed a dual color CaspaseTracker system for Drosophila to identify cells with ongoing or past caspase activity. Transient exposure of healthy females to environmental stresses such as cold shock or starvation activated the CaspaseTracker coincident with caspase activity and apoptotic morphologies in multiple cell types of developing egg chambers. Importantly, when stressed flies were returned to normal conditions, morphologically healthy egg chambers and new progeny flies were labeled by the biosensor, suggesting functional recovery from apoptotic caspase activation. In striking contrast to developing egg chambers, which lack basal caspase biosensor activation under normal conditions, many adult tissues of normal healthy flies exhibit robust caspase biosensor activity in a portion of cells, including neurons. The widespread persistence of CaspaseTracker-positivity implies that healthy cells utilize active caspases for non-apoptotic physiological functions during and after normal development.

Effect of semolina-jaggery diet on survival and development of Drosophila melanogaster

Drosophila melanogaster is an ideal model organism for developmental studies. This study tests the potential of semolina-jaggery (SJ) diet as a new formulation for bulk rearing of flies. Semolina and jaggery are organic products obtained from wheat endosperm and cane sugar, respectively. Semolina is a rich source of carbohydrates and protein. Jaggery has a high content of dietary sugars. Moreover, preparation of semolina jaggery diet is cost-effective and easy. Thus, the current study aimed to compare survival and developmental parameters of flies fed the SJ diet to flies fed the standard cornmeal-sugar-yeast (CSY) diet. SJ diet enhanced survival of flies without affecting fecundity; male flies showed increased resistance to starvation. A higher number of flies emerged at F2 and F3 generation when fed the SJ diet than when fed the control CSY diet. SJ diet did not increase fly body weight and lipid percentage. Therefore, SJ diet can be used for bulk rearing of healthy flies at par with the standard cornmeal-sugar-yeast diet.

Enhancement of the Musca domestica hytrosavirus infection with orally delivered reducing agents

House flies (Musca domestica L.) throughout the world are infected with the salivary gland hypertrophy virus MdSGHV (Hytrosaviridae). Although the primary route of infection is thought to be via ingestion, flies that are old enough to feed normally are resistant to infection per os, suggesting that the peritrophic matrix (PM) is a barrier to virus transmission. Histological examination of the peritrophic matrix of healthy flies revealed a multilaminate structure produced by midgut cells located near the proventriculus. SEM revealed the PM to form a confluent sheet surrounding the food bolus with pores/openings less than 10nm in diameter. TEM revealed the PM to be multilayered, varying in width from 350 to 900nm, and generally thinner in male than in female flies. When flies were fed on the reducing agents dithiothetriol (DTT) or tris (2-caboxyethyl)phosphine hydrochloride (TCEP) for 48h before per os exposure to the virus, infection rates increased 10- to 20-fold compared with flies that did not receive the reducing agent treatments. PM’s from flies treated with DTT and TCEP displayed varying degrees of disruption, particularly in the outer layer, and lacked the electron-dense inner layer facing the ectoperitrophic space. Both drugs were somewhat toxic to the flies, resulting in>40% mortality at doses greater than 10mM (DTT) or 5mM (TCEP). DTT increased male fly susceptibility (55.1% infected) more than that of females (7.8%), whereas TCEP increased susceptibility of females (42.9%) more than that of males (26.2%). The cause for the sex differences in response to oral exposure the reducing agents is unclear. Exposing flies to food treated with virus and the reducing agents at the same time, rather than pretreating flies with the drugs, had no effect on susceptibility to the virus. Presumably, the reducing agent disrupted the enveloped virus and acted as a viricidal agent. In summary, it is proposed that the reducing agents influence integrity of the PM barrier and increase the susceptibility of flies to infection by MdSGHV.

MEF2 Is an In Vivo Immune-Metabolic Switch

SummaryInfections disturb metabolic homeostasis in many contexts, but the underlying connections are not completely understood. To address this, we use paired genetic and computational screens in Drosophila to identify transcriptional regulators of immunity and pathology and their associated target genes and physiologies. We show that Mef2 is required in the fat body for anabolic function and the immune response. Using genetic and biochemical approaches, we find that MEF2 is phosphorylated at a conserved site in healthy flies and promotes expression of lipogenic and glycogenic enzymes. Upon infection, this phosphorylation is lost, and the activity of MEF2 changes—MEF2 now associates with the TATA binding protein to bind a distinct TATA box sequence and promote antimicrobial peptide expression. The loss of phosphorylated MEF2 contributes to loss of anabolic enzyme expression in Gram-negative bacterial infection. MEF2 is thus a critical transcriptional switch in the adult fat body between metabolism and immunity.

The Oct1 homolog Nubbin is a repressor of NF-κB-dependent immune gene expression that increases the tolerance to gut microbiota

BackgroundInnate immune responses are evolutionarily conserved processes that provide crucial protection against invading organisms. Gene activation by potent NF-κB transcription factors is essential both in mammals and Drosophila during infection and stress challenges. If not strictly controlled, this potent defense system can activate autoimmune and inflammatory stress reactions, with deleterious consequences for the organism. Negative regulation to prevent gene activation in healthy organisms, in the presence of the commensal gut flora, is however not well understood.ResultsWe show that the Drosophila homolog of mammalian Oct1/POU2F1 transcription factor, called Nubbin (Nub), is a repressor of NF-κB/Relish-driven antimicrobial peptide gene expression in flies. In nub1 mutants, which lack Nub-PD protein, excessive expression of antimicrobial peptide genes occurs in the absence of infection, leading to a significant reduction of the numbers of cultivatable gut commensal bacteria. This aberrant immune gene expression was effectively blocked by expression of Nub from a transgene. We have identified an upstream regulatory region, containing a cluster of octamer sites, which is required for repression of antimicrobial peptide gene expression in healthy flies. Chromatin immunoprecipitation experiments demonstrated that Nub binds to octamer-containing promoter fragments of several immune genes. Gene expression profiling revealed that Drosophila Nub negatively regulates many genes that are involved in immune and stress responses, while it is a positive regulator of genes involved in differentiation and metabolism.ConclusionsThis study demonstrates that a large number of genes that are activated by NF-κB/Relish in response to infection are normally repressed by the evolutionarily conserved Oct/POU transcription factor Nub. This prevents uncontrolled gene activation and supports the existence of a normal gut flora. We suggest that Nub protein plays an ancient role, shared with mammalian Oct/POU transcription factors, to moderate responses to immune challenge, thereby increasing the tolerance to biotic stress.