Accurate quantification of spatial distances between fluorescent signals in multi-channel 3D microscopy is essential for understanding genomic organization and gene regulation. However, chromatic aberration introduces systematic spatial offsets between channels that significantly bias distance measurements, particularly at short genomic distances. We present FISH-Dist, an automated computational pipeline for quantitative distance measurements in 3D fluorescence in situ hybridization (FISH) experiments acquired on standard confocal microscopes. Our method combines deep learning-based spot segmentation, 3D Gaussian fitting for sub-pixel localization, and two complementary chromatic aberration correction approaches: affine (ACC) and linear (LCC). We validated the pipeline by measuring the lengths of DNA origami nanorulers and systematically evaluated FISH probe design parameters, including probe spacing, density, and target sequence length. FISH-Dist achieves sub-pixel accuracy in signal detection and substantially reduces inter-channel distance measurement errors. This enables a reproducible quantification of spatial relationships in 3D FISH datasets. Unlike existing tools optimized for long-range chromosomal interactions or requiring super-resolution microscopy, FISH-Dist specifically addresses the technical challenges of standard confocal imaging at short genomic distances, where chromatic aberration has a proportionally greater impact on measurement accuracy.

Behavior evolution can promote the emergence of agricultural pests by changing their ecological niche. For example, the insect pest Drosophila suzukii has shifted its oviposition (egg-laying) niche from fermented fruits to ripe, non-fermented fruits, causing significant damage to a wide range of fruit crops worldwide. We investigate the chemosensory changes underlying this evolutionary shift and ask whether fruit sugars, which are depleted during fermentation, are important gustatory cues that direct D. suzukii oviposition to sweet, ripe fruits. We show that D. suzukii has expanded its range of oviposition responses to lower sugar concentrations than the model D. melanogaster, which prefers to lay eggs on fermented fruit. The increased response of D. suzukii to sugar correlates with an increase in the value of sugar relative to a fermented strawberry substrate in oviposition decisions. In addition, we show by genetic manipulation of sugar-gustatory receptor neurons (GRNs) that sugar perception is required for D. suzukii to prefer a ripe substrate over a fermented substrate, but not for D. melanogaster to prefer the fermented substrate. Thus, sugar is a major determinant of D. suzukii's choice of complex substrates. Calcium imaging experiments in the brain's primary gustatory center (suboesophageal zone) show that D. suzukii GRNs are not more sensitive to sugar than their D. melanogaster counterparts, suggesting that increased sugar valuation is encoded in downstream circuits of the central nervous system (CNS). Taken together, our data suggest that evolutionary changes in central brain sugar valuation computations are involved in driving D. suzukii's oviposition preference for sweet, ripe fruit.

Behavior evolution can promote the emergence of agricultural pests via ecological niche changes. The underlying neuronal mechanisms are poorly understood. We investigate the chemosensory changes underlying the evolutionary shift of oviposition substrate of the pest Drosophila suzukii from rotten to ripe fruits. Using a model substrate for fermented fruits and genetic manipulations, we show that an increase in valuation of fruit sugars during oviposition decisions drives D. suzukii to oviposit on ripe fruits as opposed to the model D. melanogaster which prefers rotten fruits. Inter-species comparative in vivo calcium imaging of sugar Gustatory Receptor Neurons suggests that increased sugar valuation in D. suzukii is related to neuronal sensitivity changes at multiple levels of the oviposition circuitry. Our results show that the tuning of sugar valuation has contributed to the evolution of oviposition preference on ripe fruit of D. suzukii .

Bacteria that colonize eukaryotic gut have profound influences on the physiology of their host. In Drosophila, many of these effects are mediated by adipocytes that combine immune and metabolic functions. We show here that enteric infection with some bacteria species triggers the activation of the SREBP lipogenic protein in surrounding enterocytes but also in remote fat body cells and in ovaries, an effect that requires insulin signaling. We demonstrate that by activating the NF-κB pathway, the cell wall peptidoglycan produced by the same gut bacteria remotely, and cell-autonomously, represses SREBP activation in adipocytes. We finally show that by reducing the level of peptidoglycan, the gut born PGRP-LB amidase balances host immune and metabolic responses of the fat body to gut-associated bacteria. In the absence of such modulation, uncontrolled immune pathway activation prevents SREBP activation and lipid production by the fat body.

Bacteria that colonize eukaryotic gut have profound influences on the physiology of their host. In Drosophila, many of these effects are mediated by the adipocytes that combine immune and metabolic functions. We show here that gut colonization by specific bacteria species stimulate lipogenesis in surrounding enterocytes but also in remote fat body cells and ovaries. This bacteria-dependent lipid production is mediated by SREBP and requires a functional insulin signaling. However, it is antagonized by microbiota-born peptidoglycan which by activating NF-κB signaling, cell-autonomously represses SREPP activation in adipocytes but not in enterocytes. We finally show that by reducing microbiota-derived PGN, the gut-produced PGRP-LB amidase balances host immune and metabolic responses of the fat body to gut-associated bacteria. In the absence of such modulation, uncontrolled immune pathway activation prevents lipid production by the fat body resulting in infection-dependent host death. Bullets Gut microbiota activates lipogenesis locally in enterocytes and remotely in adipocytes Bacteria-dependent activation of SREBP in adipocytes is mediated by insulin signaling Activation of lipid synthesis by microbiota is antagonized by NF-κB signaling triggered by gut-born peptidoglycan By reducing peptidoglycan circulating levels, PGRP-LB maintains a balance between immune and metabolic response to microbiota

The phytopathogen Erwinia carotovora carotovora (Ecc) has been used successfully to decipher some of the mechanisms that regulate the interactions between Drosophila melanogaster and bacteria, mostly following forced association between the two species. How do Drosophila normally perceive and respond to the presence of Ecc is unknown. Using a fly feeding two-choice assay and video tracking, we show that Drosophila are first attracted but then repulsed by an Ecc-contaminated solution. The initial attractive phase is dependent on the olfactory Gr63a and Gαq proteins, whereas the second repulsive phase requires a functional gustatory system. Genetic manipulations and calcium imaging indicate that bitter neurons and gustatory receptors Gr66a and Gr33a are needed for the aversive phase and that the neuropeptide leukokinin is also involved. We also demonstrate that these behaviors are independent of the NF-κB cascade that controls some of the immune, metabolic, and behavioral responses to bacteria.

This protocol is designed to prepare adult axenic Drosophila before monitoring their behavior in a two-choice feeding assay, where flies are confronted with an axenic versus a dead or alive bacteria-contaminated feeding solution. Several aspects of the procedure, including raising and aging flies in axenic conditions, starving adult flies, and composing feeding solutions, are detailed. The bacterium used in this protocol, Erwinia carotovora carotovora-152141 (Ecc-152141), is commonly used to decipher the mechanisms controlling host-pathogen interactions in the Drosophila model. For complete details on the use and execution of this protocol, please refer to Charroux et al. (2020).

Gut-associated bacteria produce metabolites that both have a local influence on the intestinal tract and act at a distance on remote organs. In Drosophila, bacteria-derived peptidoglycan (PGN) displays such a dual role. PGN triggers local antimicrobial peptide production by enterocytes; it also activates systemic immune responses in fat-body cells and modulates fly behavior by acting on neurons. How these responses to a single microbiota-derived compound are simultaneously coordinated is not understood. We show here that the PGRP-LB locus generates both cytosolic and secreted PGN-cleaving enzymes. Through genetic analysis, we demonstrate that the cytosolic PGRP-LB isoforms cell-autonomously control the intensity of NF-κB activation in enterocytes, whereas the secreted isoform prevents massive and detrimental gut-derived PGN dissemination throughout the organism. This study explains how Drosophila are able to uncouple the modulation of local versus systemic responses to a single gut-bacteria-derived product by using isoform-specific enzymes.

NF-κB pathways are key signaling cascades of the Drosophila innate immune response. One of them, the Immune Deficiency (IMD) pathway, is under a very tight negative control. Although molecular brakes exist at each step of this signaling module from ligand availability to transcriptional regulation, it remains unknown whether repressors act in the same cells or tissues and if not, what is rationale behind this spatial specificity. We show here that the negative regulator of IMD pathway PGRP-LF is epressed in ectodermal derivatives. We provide evidence that, in the absence of any immune elicitor, PGRP-LF loss-of-function mutants, display a constitutive NF-κB/IMD activation specifically in ectodermal tissues leading to genitalia and tergite malformations. In agreement with previous data showing that proper development of these structures requires induction of apoptosis, we show that ectopic activation of NF-κB/IMD signaling leads to apoptosis inhibition in both genitalia and tergite primordia. We demonstrate that NF-κB/IMD signaling antagonizes apoptosis by up-regulating expression of the anti-apoptotic protein Diap1. Altogether these results show that, in the complete absence of infection, the negative regulation of NF-κB/IMD pathway by PGRP-LF is crucial to ensure proper induction of apoptosis and consequently normal fly development. These results highlight that IMD pathway regulation is controlled independently in different tissues, probably reflecting the different roles of this signaling cascade in both developmental and immune processes.

As infectious diseases pose a threat to host integrity, eukaryotes have evolved mechanisms to eliminate pathogens. In addition to develop strategies reducing infection, animals can engage in behaviors that lower the impact of the infection. The molecular mechanisms by which microbes impact host behavior are not well understood. We demonstrate that bacterial infection of Drosophila females reduces oviposition and that peptidoglycan, the component that activates Drosophila antibacterial response, is also the elicitor of this behavioral change. We show that peptidoglycan regulates egg-laying rate by activating NF-κB signaling pathway in octopaminergic neurons and that, a dedicated peptidoglycan degrading enzyme acts in these neurons to buffer this behavioral response. This study shows that a unique ligand and signaling cascade are used in immune cells to mount an immune response and in neurons to control fly behavior following infection. This may represent a case of behavioral immunity.

Peptidoglycan (PGN) detection by PGN recognition proteins (PGRP) is the main trigger of the antibacterial immune response in Drosophila. Depending on the type of immune cell, PGN can be sensed either at the cell membrane by PGRP-LC or inside the cell by PGRP-LE, which plays a role similar to that of Nod2 in mammals. Previous work, mainly in cell cultures, has shown that oligopeptide transporters of the SLC15 family are essential for the delivery of PGN for Nod2 detection inside of the cells, and that this function might be conserved in flies. By generating and analyzing the immune phenotypes of loss-of-function mutations in 3 SLC15 Drosophila family members, we tested their role in mediating PGRP-LE-dependent PGN activation. Our results show that Yin, CG2930, and CG9444 are required neither for PGRP-LE activation by PGN nor for PGN transport from the gut lumen to the insect blood. These data show that, while intracellular PGN detection is an essential step of the antibacterial response in both insects and mammals, the types of PGN transporters and sensors are different in these animals.

In Drosophila, peptidoglycan (PGN) is detected by PGN recognition proteins (PGRPs) that act as pattern recognition receptors. Some PGRPs such as PGRP-LB or PGRP-SCs are able to cleave PGN, therefore reducing the amount of immune elicitors and dampening immune deficiency (IMD) pathway activation. The precise role of PGRP-SC is less well defined because the PGRP-SC genes (PGRP-SC1a, PGRP-SC1b and PGRP-SC2) lie very close on the chromosome and have been studied using a deletion encompassing the three genes. By generating PGRP-SC-specific mutants, we reevaluated the roles of PGRP-LB, PGRP-SC1 and PGRP-SC2, respectively, during immune responses. We showed that these genes are expressed in different gut domains and that they follow distinct transcriptional regulation. Loss-of-function mutant analysis indicates that PGRP-LB is playing a major role in IMD pathway activation and bacterial load regulation in the gut, although PGRP-SCs are expressed at high levels in this organ. We also demonstrated that PGRP-SC2 is the main negative regulator of IMD pathway activation in the fat body. Accordingly, we showed that mutants for either PGRP-LB or PGRP-SC2 displayed a distinct susceptibility to bacteria depending on the infection route. Lastly, we demonstrated that PGRP-SC1 and PGRP-SC2 are required in vivo for full Toll pathway activation by Gram-positive bacteria.

All insects are colonized by microorganisms on their exoskeleton, their gut and even in some cases within their own somatic and germ line cells. This microbiota that can represent up to a few percent of the insect biomass may have a pervasive impact on many aspects of insect biology including physiology, nutrient acquisition, ageing, behaviour and resistance to infection. Mainly through ingestion of contaminated food, the mouth-gut axis represents the first and principal access of external bacteria to the host. Soon after ingestion, the feeding insect needs to rapidly and accurately identify the ingested microbes and decide whether to preserve them if beneficial or neutral, or to eliminate them if potentially harmful. We will review here the recent data acquired in Drosophila on the mechanisms that invertebrate enterocytes rely on to detect the presence of bacteria in the gut. We will compare these modes of bacteria sensing to those in other immune competent tissues and try to rationalize differences that may exist. We will also analyse the physiological consequences of bacteria detection not only locally for the gut itself but also for remote tissues. Finally, we will describe the physiological disorders that can occur due to inaccurate bacteria identification by the gut epithelium.

In Drosophila, peptidoglycan (PGN) is detected by PGN recognition proteins (PGRPs) that act as pattern recognition receptors. Some PGRPs such as PGRP-LB or PGRP-SCs are able to cleave PGN, therefore reducing the amount of immune elicitors and dampening immune deficiency (IMD) pathway activation. The precise role of PGRP-SC is less well defined because the PGRP-SC genes (PGRP-SC1a, PGRP-SC1b and PGRP-SC2) lie very close on the chromosome and have been studied using a deletion encompassing the three genes. By generating PGRP-SC-specific mutants, we reevaluated the roles of PGRP-LB, PGRP-SC1 and PGRP-SC2, respectively, during immune responses. We showed that these genes are expressed in different gut domains and that they follow distinct transcriptional regulation. Loss-of-function mutant analysis indicates that PGRP-LB is playing a major role in IMD pathway activation and bacterial load regulation in the gut, although PGRP-SCs are expressed at high levels in this organ. We also demonstrated that PGRP-SC2 is the main negative regulator of IMD pathway activation in the fat body. Accordingly, we showed that mutants for either PGRP-LB or PGRP-SC2 displayed a distinct susceptibility to bacteria depending on the infection route. Lastly, we demonstrated that PGRP-SC1 and PGRP-SC2 are required in vivo for full Toll pathway activation by Gram-positive bacteria.

Summary Sorting of secretory cargo and retrieval of components of the biosynthetic pathway occur in organelles such as the Golgi apparatus, the endoplasmic reticulum and the endosomes. In order to perform their functions in protein sorting, these organelles require a weakly acidified lumen. In vitro data have shown that Golgi luminal pH is in part regulated by an anion channel called Golgi pH Regulator (GPHR). Mammalian cells carrying a mutated GPHR version present an increased luminal pH leading to delayed protein transport, impaired glycosylation and Golgi disorganization. Using Drosophila as a model system, we present here the first phenotypic consequences, at the organism level, of a complete lack of GPHR function. We show that, although all individuals carrying complete loss-of-function mutations in the dGPHR gene can go through embryonic development, most of them die at late larval stages. The dGPHR mutations are, however, sublethal and can therefore generate escapers that are smaller than controls. Using cellular and molecular readouts, we demonstrate that the effects of dGPHR mutation on larval growth are not due to Insulin signaling pathway impairment and can be rescued by providing dGPHR in only some of the larval tissues. We reveal that, although functionally exchangeable, the invertebrate and vertebrate GPHRs display not completely overlapping sub-cellular localization. Whereas the mammalian GPHR is a Golgi-only associated protein whose inactivation disturbs the Golgi apparatus, our data suggest that dGPHR is expressed in both the ER and the Golgi and that dGPHR mutant flies have defects in both organelles that lead to a defective secretory pathway.

Since insect mostly developed on decaying matter and contaminated fruits, they are constantly ingesting bacteria. The insect model, Drosophila, is therefore well adapted to study the interactions that take place between the gut epithelia and either resident or infectious bacteria. In order to provide an ad hoc immune response, gut epithelial cells must detect the presence of bacteria. In a recent report, Bosco-Drayon et al. identify the main receptors by which Drosophila sense gut associated bacteria and analyze how this bacteria-receptor interaction translate into gene activation.

Gut epithelial cells contact both commensal and pathogenic bacteria, and proper responses to these bacteria require a balance of positive and negative regulatory signals. In the Drosophila intestine, peptidoglycan-recognition proteins (PGRPs), including PGRP-LE, play central roles in bacterial recognition and activation of immune responses, including induction of the IMD-NF-κB pathway. We show that bacteria recognition is regionalized in the Drosophila gut with various functional regions requiring different PGRPs. Specifically, peptidoglycan recognition by PGRP-LE in the gut induces NF-κB-dependent responses to infectious bacteria but also immune tolerance to microbiota through upregulation of pirk and PGRP-LB, which negatively regulate IMD pathway activation. Loss of PGRP-LE-mediated detection of bacteria in the gut results in systemic immune activation, which can be rescued by overexpressing PGRP-LB in the gut. Together these data indicate that PGRP-LE functions as a master gut bacterial sensor that induces balanced responses to infectious bacteria and tolerance to microbiota.

Syndromic diarrhea (or trichohepatoenteric syndrome) is a rare congenital bowel disorder characterized by intractable diarrhea and woolly hair, and it has recently been associated with mutations in TTC37. Although databases report TTC37 as being the human ortholog of Ski3p, one of the yeast Ski-complex cofactors, this lead was not investigated in initial studies. The Ski complex is a multiprotein complex required for exosome-mediated RNA surveillance, including the regulation of normal mRNA and the decay of nonfunctional mRNA. Considering the fact that TTC37 is homologous to Ski3p, we explored a gene encoding another Ski-complex cofactor, SKIV2L, in six individuals presenting with typical syndromic diarrhea without variation in TTC37. We identified mutations in all six individuals. Our results show that mutations in genes encoding cofactors of the human Ski complex cause syndromic diarrhea, establishing a link between defects of the human exosome complex and a Mendelian disease.

Millions of people suffer from inflammatory diseases of the intestine, some of them potentiating gastrointestinal cancer. These gut-associated pathologies arise from imbalanced interactions between the host gut epithelia and resident or ingested microbes, interactions that are still poorly understood at the molecular level. Drosophila has been a very powerful model to study development and diseases. Its relatively simple tissue organization and sophisticated genetics are some of the advantages of using it as an experimental model to dissect gut-microbe interactions. Recent progress made in various research fields such as Drosophila microbiota composition, gut epithelium structure or gut immune reactions led us to believe that Drosophila is becoming an ad hoc model system to dissect the mechanisms that cooperate to maintain intestinal homeostasis in higher eukaryotes. It further may help us understand how an alteration of these finely tuned processes precipitates the inflammatory processes found in some inflammatory bowel diseases.