Recognition of nonself is necessary to activate Drosophila's immune response against an insect parasite.

BACKGROUND: Innate immune responses can be activated by pathogen-associated molecular patterns (PAMPs), danger signals released by damaged tissues, or the absence of self-molecules that inhibit immunity. As PAMPs are typically conserved across broad groups of pathogens but absent from the host, it is unclear whether they allow hosts to recognize parasites that are phylogenetically similar to themselves, such as parasitoid wasps infecting insects. RESULTS: Parasitoids must penetrate the cuticle of Drosophila larvae to inject their eggs. In line with previous results, we found that the danger signal of wounding triggers the differentiation of specialized immune cells called lamellocytes. However, using oil droplets to mimic infection by a parasitoid wasp egg, we found that this does not activate the melanization response. This aspect of the immune response also requires exposure to parasite molecules. The unidentified factor enhances the transcriptional response in hemocytes and induces a specific response in the fat body. CONCLUSIONS: We conclude that a combination of danger signals and the recognition of nonself molecules is required to activate Drosophila's immune response against parasitic insects.

A PI3K-calcium-Nox axis primes leukocyte Nrf2 to boost immune resilience and limit collateral damage.

Phagosomal reactive oxygen species (ROS) are strategically employed by leukocytes to kill internalized pathogens and degrade cellular debris. Nevertheless, uncontrolled oxidant bursts could cause serious collateral damage to phagocytes or other host tissues, potentially accelerating aging and compromising host viability. Immune cells must, therefore, activate robust self-protective programs to mitigate these undesired effects, and yet allow crucial cellular redox signaling. Here, we dissect in vivo the molecular nature of these self-protective pathways, their precise mode of activation, and physiological effects. We reveal Drosophila embryonic macrophages activate the redox-sensitive transcription factor Nrf2 upon corpse engulfment during immune surveillance, downstream of calcium- and PI3K-dependent ROS release by phagosomal Nox. By transcriptionally activating the antioxidant response, Nrf2 not only curbs oxidative damage but preserves vital immune functions (including inflammatory migration) and delays the acquisition of senescence-like features. Strikingly, macrophage Nrf2 also acts non-autonomously to limit ROS-induced collateral damage to surrounding tissues. Cytoprotective strategies may thus offer powerful therapeutic opportunities for alleviating inflammatory or age-related diseases.

Interaction of lncRNA-CR33942 with Dif/Dorsal Facilitates Antimicrobial Peptide Transcriptions and Enhances Drosophila Toll Immune Responses.

The Drosophila Toll signaling pathway mainly responds to Gram-positive (G+) bacteria or fungal infection, which is highly conserved with mammalian TLR signaling pathway. Although many positive and negative regulators involved in the immune response of the Toll pathway have been identified in Drosophila, the roles of long noncoding RNAs (lncRNAs) in Drosophila Toll immune responses are poorly understood to date. In this study, our results demonstrate that lncRNA-CR33942 is mainly expressed in the nucleus and upregulated after Micrococcus luteus infection. Especially, lncRNA-CR33942 not only modulates differential expressions of multiple antimicrobial peptide genes but also affects the Drosophila survival rate during response to G+ bacterial infection based on the transiently overexpressing and the knockdown lncRNA-CR33942 assays in vivo. Mechanically, lncRNA-CR33942 interacts with the NF-κB transcription factors Dorsal-related immunity factor/Dorsal to promote the transcriptions of antimicrobial peptides drosomycin and metchnikowin, thus enhancing Drosophila Toll immune responses. Taken together, this study identifies lncRNA-CR33942 as a positive regulator of Drosophila innate immune response to G+ bacterial infection to facilitate Toll signaling via interacting with Dorsal-related immunity factor/Dorsal. It would be helpful to reveal the roles of lncRNAs in Toll immune response in Drosophila and provide insights into animal innate immunity.

Roles of matrix metalloproteinases in development, immunology, and ovulation in fruit Fly (Drosophila).

Matrix metalloproteinase (MMP), a protease enzyme, participates in proteolytic cleavage of extracellular matrix proteins from Drosophila and mammals. But, recent studies have revealed other physiologically important roles of MMP in Drosophila. MMP contributes to cardioblast movement and distribution of collagen proteins during cardiogenesis in developing Drosophila. Tissue remodeling, especially tracheal development is also maintained by MMP. MMP regulates certain immunological functions in Drosophila such as wound repairing, plasmatocyte assemblage at the injured site of the basement membrane and glial response to axon degeneration in Drosophila nervous system. But, the contribution of MMP to tumor formation and metastasis in Drosophila has made it an interesting topic among researchers. Ovulation and egg laying are also found to be affected positively by MMP in Drosophila.

Male Age and Wolbachia Dynamics: Investigating How Fast and Why Bacterial Densities and Cytoplasmic Incompatibility Strengths Vary.

Endosymbionts can influence host reproduction and fitness to favor their maternal transmission. For example, endosymbiotic Wolbachia bacteria often cause cytoplasmic incompatibility (CI) that kills uninfected embryos fertilized by Wolbachia-modified sperm. Infected females can rescue CI, providing them a relative fitness advantage. Wolbachia-induced CI strength varies widely and tends to decrease as host males age. Since strong CI drives Wolbachia to high equilibrium frequencies, understanding how fast and why CI strength declines with male age is crucial to explaining age-dependent CI's influence on Wolbachia prevalence. Here, we investigate if Wolbachia densities and/or CI gene (cif) expression covary with CI-strength variation and explore covariates of age-dependent Wolbachia-density variation in two classic CI systems. wRi CI strength decreases slowly with Drosophila simulans male age (6%/day), but wMel CI strength decreases very rapidly (19%/day), yielding statistically insignificant CI after only 3 days of Drosophila melanogaster adult emergence. Wolbachia densities and cif expression in testes decrease as wRi-infected males age, but both surprisingly increase as wMel-infected males age, and CI strength declines. We then tested if phage lysis, Octomom copy number (which impacts wMel density), or host immune expression covary with age-dependent wMel densities. Only host immune expression correlated with density. Together, our results identify how fast CI strength declines with male age in two model systems and reveal unique relationships between male age, Wolbachia densities, cif expression, and host immunity. We discuss new hypotheses about the basis of age-dependent CI strength and its contributions to Wolbachia prevalence. IMPORTANCEWolbachia bacteria are the most common animal-associated endosymbionts due in large part to their manipulation of host reproduction. Many Wolbachia cause cytoplasmic incompatibility (CI) that kills uninfected host eggs. Infected eggs are protected from CI, favoring Wolbachia spread in natural systems and in transinfected mosquito populations where vector-control groups use strong CI to maintain pathogen-blocking Wolbachia at high frequencies for biocontrol of arboviruses. CI strength varies considerably in nature and declines as males age for unknown reasons. Here, we determine that CI strength weakens at different rates with age in two model symbioses. Wolbachia density and CI gene expression covary with wRi-induced CI strength in Drosophila simulans, but neither explain rapidly declining wMel-induced CI in aging D. melanogaster males. Patterns of host immune gene expression suggest a candidate mechanism behind age-dependent wMel densities. These findings inform how age-dependent CI may contribute to Wolbachia prevalence in natural systems and potentially in transinfected systems.

The Cellular Innate Immune Response of the Invasive Pest Insect Drosophila suzukii against Pseudomonas entomophila Involves the Release of Extracellular Traps.

Drosophila suzukii is a neobiotic invasive pest that causes extensive damage to fruit crops worldwide. The biological control of this species has been unsuccessful thus far, in part because of its robust cellular innate immune system, including the activity of professional phagocytes known as hemocytes and plasmatocytes. The in vitro cultivation of primary hemocytes isolated from D. suzukii third-instar larvae is a valuable tool for the investigation of hemocyte-derived effector mechanisms against pathogens such as wasp parasitoid larvae, bacteria, fungi and viruses. Here, we describe the morphological characteristics of D. suzukii hemocytes and evaluate early innate immune responses, including extracellular traps released against the entomopathogen Pseudomonas entomophila and lipopolysaccharides. We show for the first time that D. suzukii plasmatocytes cast extracellular traps to combat P. entomophila, along with other cell-mediated reactions, such as phagocytosis and the formation of filopodia.

Two oppositely-charged sf3b1 mutations cause defective development, impaired immune response, and aberrant selection of intronic branch sites in Drosophila.

SF3B1 mutations occur in many cancers, and the highly conserved His662 residue is one of the hotspot mutation sites. To address effects on splicing and development, we constructed strains carrying point mutations at the corresponding residue His698 in Drosophila using the CRISPR-Cas9 technique. Two mutations, H698D and H698R, were selected due to their frequent presence in patients and notable opposite charges. Both the sf3b1-H698D and-H698R mutant flies exhibit developmental defects, including less egg-laying, decreased hatching rates, delayed morphogenesis and shorter lifespans. Interestingly, the H698D mutant has decreased resistance to fungal infection, while the H698R mutant shows impaired climbing ability. Consistent with these phenotypes, further analysis of RNA-seq data finds altered expression of immune response genes and changed alternative splicing of muscle and neural-related genes in the two mutants, respectively. Expression of Mef2-RB, an isoform of Mef2 gene that was downregulated due to splicing changes caused by H698R, partly rescues the climbing defects of the sf3b1-H698R mutant. Lariat sequencing reveals that the two sf3b1-H698 mutations cause aberrant selection of multiple intronic branch sites, with the H698R mutant using far upstream branch sites in the changed alternative splicing events. This study provides in vivo evidence from Drosophila that elucidates how these SF3B1 hotspot mutations alter splicing and their consequences in development and in the immune system.

Downregulation of Perilipin1 by the Immune Deficiency Pathway Leads to Lipid Droplet Reconfiguration and Adaptation to Bacterial Infection in Drosophila.

Lipid droplets (LDs), the highly dynamic intracellular organelles, are critical for lipid metabolism. Dynamic alterations in the configurations and functions of LDs during innate immune responses to bacterial infections and the underlying mechanisms, however, remain largely unknown. In this study, we trace the time-course morphology of LDs in fat bodies of Drosophila after transient bacterial infection. Detailed analysis shows that perilipin1 (plin1), a core gene involved in the regulation of LDs, is suppressed by the immune deficiency signaling, one major innate immune pathway in Drosophila During immune activation, downregulated plin1 promotes the enlargement of LDs, which in turn alleviates immune reaction-associated reactive oxygen species stress. Thus, the growth of LDs is likely an active adaptation to maintain redox homeostasis in response to immune deficiency activation. Therefore, our study provides evidence that plin1 serves as a modulator on LDs' reconfiguration in regulating infection-induced pathogenesis, and plin1 might be a potential therapeutic target for coordinating inflammation resolution and lipid metabolism.

Impact of Microorganisms and Parasites on Neuronally Controlled Drosophila Behaviours.

Like all invertebrates, flies such as Drosophila lack an adaptive immune system and depend on their innate immune system to protect them against pathogenic microorganisms and parasites. In recent years, it appears that the nervous systems of eucaryotes not only control animal behavior but also cooperate and synergize very strongly with the animals' immune systems to detect and fight potential pathogenic threats, and allow them to adapt their behavior to the presence of microorganisms and parasites that coexist with them. This review puts into perspective the latest progress made using the Drosophila model system, in this field of research, which remains in its infancy.

Circadian regulation of the Drosophila astrocyte transcriptome.

Recent studies have demonstrated that astrocytes cooperate with neurons of the brain to mediate circadian control of many rhythmic processes including locomotor activity and sleep. Transcriptional profiling studies have described the overall rhythmic landscape of the brain, but few have employed approaches that reveal heterogeneous, cell-type specific rhythms of the brain. Using cell-specific isolation of ribosome-bound RNAs in Drosophila, we constructed the first circadian "translatome" for astrocytes. This analysis identified 293 "cycling genes" in astrocytes, most with mammalian orthologs. A subsequent behavioral genetic screen identified a number of genes whose expression is required in astrocytes for normal sleep behavior. In particular, we show that certain genes known to regulate fly innate immune responses are also required for normal sleep patterns.

Two sequential activation modules control the differentiation of protective T helper-1 (Th1) cells.

Interferon-γ (IFN-γ)-producing CD4+ T helper-1 (Th1) cells are critical for protection from microbes that infect the phagosomes of myeloid cells. Current understanding of Th1 cell differentiation is based largely on reductionist cell culture experiments. We assessed Th1 cell generation in vivo by studying antigen-specific CD4+ T cells during infection with the phagosomal pathogen Salmonella enterica (Se), or influenza A virus (IAV), for which CD4+ T cells are less important. Both microbes induced T follicular helper (Tfh) and interleukin-12 (IL-12)-independent Th1 cells. During Se infection, however, the Th1 cells subsequently outgrew the Tfh cells via an IL-12-dependent process and formed subsets with increased IFN-γ production, ZEB2-transcription factor-dependent cytotoxicity, and capacity to control Se infection. Our results indicate that many infections induce a module that generates Tfh and poorly differentiated Th1 cells, which is followed in phagosomal infections by an IL-12-dependent Th1 cell amplification module that is critical for pathogen control.

Homeostatic Regulation of ROS-Triggered Hippo-Yki Pathway via Autophagic Clearance of Ref(2)P/p62 in the Drosophila Intestine.

Homeostasis of intestinal epithelia is maintained by coordination of the proper rate of regeneration by stem cell division with the rate of cell loss. Regeneration of host epithelia is normally quiescent upon colonization of commensal bacteria; however, the epithelia often develop dysplasia in a context-dependent manner, the cause and underlying mechanism of which remain unclear. Here, we show that in Drosophila intestine, autophagy lowers the sensitivity of differentiated enterocytes to reactive oxygen species (ROS) that are produced in response to commensal bacteria. We find that autophagy deficiency provokes ROS-dependent excessive regeneration and subsequent epithelial dysplasia and barrier dysfunction. Mechanistically, autophagic substrate Ref(2)P/p62, which co-localizes and physically interacts with Dachs, a Hippo signaling regulator, accumulates upon autophagy deficiency and thus inactivates Hippo signaling, resulting in stem cell over-proliferation non-cell autonomously. Our findings uncover a mechanism whereby suppression of undesirable regeneration by autophagy maintains long-term homeostasis of intestinal epithelia.

Cellular and humoral immune interactions between Drosophila and its parasitoids.

The immune interactions occurring between parasitoids and their host insects, especially in Drosophila-wasp models, have long been the research focus of insect immunology and parasitology. Parasitoid infestation in Drosophila is counteracted by its multiple natural immune defense systems, which include cellular and humoral immunity. Occurring in the hemocoel, cellular immune responses involve the proliferation, differentiation, migration and spreading of host hemocytes and parasitoid encapsulation by them. Contrastingly, humoral immune responses rely more heavily on melanization and on the Toll, Imd and Jak/Stat immune pathways associated with antimicrobial peptides along with stress factors. On the wasps' side, successful development is achieved by introducing various virulence factors to counteract immune responses of Drosophila. Some or all of these factors manipulate the host's immunity for successful parasitism. Here we review current knowledge of the cellular and humoral immune interactions between Drosophila and its parasitoids, focusing on the defense mechanisms used by Drosophila and the strategies evolved by parasitic wasps to outwit it.

Metabolic control of cellular immune-competency by odors in Drosophila.

Studies in different animal model systems have revealed the impact of odors on immune cells; however, any understanding on why and how odors control cellular immunity remained unclear. We find that Drosophila employ an olfactory-immune cross-talk to tune a specific cell type, the lamellocytes, from hematopoietic-progenitor cells. We show that neuronally released GABA derived upon olfactory stimulation is utilized by blood-progenitor cells as a metabolite and through its catabolism, these cells stabilize Sima/HIFα protein. Sima capacitates blood-progenitor cells with the ability to initiate lamellocyte differentiation. This systemic axis becomes relevant for larvae dwelling in wasp-infested environments where chances of infection are high. By co-opting the olfactory route, the preconditioned animals elevate their systemic GABA levels leading to the upregulation of blood-progenitor cell Sima expression. This elevates their immune-potential and primes them to respond rapidly when infected with parasitic wasps. The present work highlights the importance of the olfaction in immunity and shows how odor detection during animal development is utilized to establish a long-range axis in the control of blood-progenitor competency and immune-priming.

Immune interactions between Drosophila and the pathogen Xenorhabdus.

Deciphering host innate immune function and bacterial pathogenic tactics require a system that facilitates both facets of host-pathogen interactions. In recent years, a model that becomes established in dissecting mechanisms of host antibacterial immune response through probing with a potent bacterial pathogen involves the fruit fly Drosophila melanogaster and the insect pathogenic bacteria Xenorhabdus spp. The elegance of this system involves not only the genetic tractability of D. melanogaster, but also the association of Xenorhabdus with parasitic nematodes of insects that supervise the release of the bacteria as well as influence their pathogenic properties during the infection process. These dynamic aspects have enabled us to start decoding the specific features of the D. melanogaster host defense that participate in confronting the activity of Xenorhabdus molecular components, which are designed to evade the immune system. Here we outline recent information on the cellular, humoral and phenoloxidase reactions that are induced in D. melanogaster larvae and adults to oppose the Xenorhabdus attack, and the bacterial factors responsible for triggering these effects. This knowledge is critical not only for understanding how invertebrate immunity operates, but also for devising novel approaches to exploit the virulence ability of certain bacteria with the ultimate goal to counteract harmful insect pests or vectors of infectious disease.

Immune Control of Animal Growth in Homeostasis and Nutritional Stress in Drosophila.

A large body of research implicates the brain and fat body (liver equivalent) as central players in coordinating growth and nutritional homeostasis in multicellular animals. In this regard, an underlying connection between immune cells and growth is also evident, although mechanistic understanding of this cross-talk is scarce. Here, we explore the importance of innate immune cells in animal growth during homeostasis and in conditions of nutrient stress. We report that Drosophila larvae lacking blood cells eclose as small adults and show signs of insulin insensitivity. Moreover, when exposed to dietary stress of a high-sucrose diet (HSD), these animals are further growth retarded than normally seen in regular animals raised on HSD. In contrast, larvae carrying increased number of activated macrophage-like plasmatocytes show no defects in adult growth when raised on HSD and grow to sizes almost comparable with that seen with regular diet. These observations imply a central role for immune cell activity in growth control. Mechanistically, our findings reveal a surprising influence of immune cells on balancing fat body inflammation and insulin signaling under conditions of homeostasis and nutrient overload as a means to coordinate systemic metabolism and adult growth. This work integrates both the cellular and humoral arm of the innate immune system in organismal growth homeostasis, the implications of which may be broadly conserved across mammalian systems as well.

A Transposon Story: From TE Content to TE Dynamic Invasion of Drosophila Genomes Using the Single-Molecule Sequencing Technology from Oxford Nanopore.

Transposable elements (TEs) are the main components of genomes. However, due to their repetitive nature, they are very difficult to study using data obtained with short-read sequencing technologies. Here, we describe an efficient pipeline to accurately recover TE insertion (TEI) sites and sequences from long reads obtained by Oxford Nanopore Technology (ONT) sequencing. With this pipeline, we could precisely describe the landscapes of the most recent TEIs in wild-type strains of Drosophila melanogaster and Drosophila simulans. Their comparison suggests that this subset of TE sequences is more similar than previously thought in these two species. The chromosome assemblies obtained using this pipeline also allowed recovering piRNA cluster sequences, which was impossible using short-read sequencing. Finally, we used our pipeline to analyze ONT sequencing data from a D. melanogaster unstable line in which LTR transposition was derepressed for 73 successive generations. We could rely on single reads to identify new insertions with intact target site duplications. Moreover, the detailed analysis of TEIs in the wild-type strains and the unstable line did not support the trap model claiming that piRNA clusters are hotspots of TE insertions.