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.

The evolution of constitutively active humoral immune defenses in Drosophila populations under high parasite pressure.

Both constitutive and inducible immune mechanisms are employed by hosts for defense against infection. Constitutive immunity allows for a faster response, but it comes with an associated cost that is always present. This trade-off between speed and fitness costs leads to the theoretical prediction that constitutive immunity will be favored where parasite exposure is frequent. We selected populations of Drosophila melanogaster under high parasite pressure from the parasitoid wasp Leptopilina boulardi. With RNA sequencing, we found the evolution of resistance in these populations was associated with them developing constitutively active humoral immunity, mediated by the larval fat body. Furthermore, these evolved populations were also able to induce gene expression in response to infection to a greater level, which indicates an overall more activated humoral immune response to parasitization. The anti-parasitoid immune response also relies on the JAK/STAT signaling pathway being activated in muscles following infection, and this induced response was only seen in populations that had evolved under high parasite pressure. We found that the cytokine Upd3, which induces this JAK/STAT response, is being expressed by immature lamellocytes. Furthermore, these immune cells became constitutively present when populations evolved resistance, potentially explaining why they gained the ability to activate JAK/STAT signaling. Thus, under intense parasitism, populations evolved resistance by increasing both constitutive and induced immune defenses, and there is likely an interplay between these two forms of immunity.

Activation of immune defences against parasitoid wasps does not underlie the cost of infection.

Parasites reduce the fitness of their hosts, and different causes of this damage have fundamentally different consequences for the evolution of immune defences. Damage to the host may result from the parasite directly harming its host, often due to the production of virulence factors that manipulate host physiology. Alternatively, the host may be harmed by the activation of its own immune defences, as these can be energetically demanding or cause self-harm. A well-studied model of the cost of infection is Drosophila melanogaster and its common natural enemy, parasitoid wasps. Infected Drosophila larvae rely on humoral and cellular immune mechanisms to form a capsule around the parasitoid egg and kill it. Infection results in a developmental delay and reduced adult body size. To disentangle the effects of virulence factors and immune defences on these costs, we artificially activated anti-parasitoid immune defences in the absence of virulence factors. Despite immune activation triggering extensive differentiation and proliferation of immune cells together with hyperglycaemia, it did not result in a developmental delay or reduced body size. We conclude that the costs of infection do not result from these aspects of the immune response and may instead result from the parasite directly damaging the host.

Natural selection has driven the recurrent loss of an immunity gene that protects Drosophila against a major natural parasite.

Polymorphisms in immunity genes can have large effects on susceptibility to infection. To understand the origins of this variation, we have investigated the genetic basis of resistance to the parasitoid wasp Leptopilina boulardi in Drosophila melanogaster. We found that increased expression of the gene lectin-24A after infection by parasitic wasps was associated with a faster cellular immune response and greatly increased rates of killing the parasite. lectin-24A encodes a protein that is strongly up-regulated in the fat body after infection and localizes to the surface of the parasite egg. In certain susceptible lines, a deletion upstream of the lectin-24A has largely abolished expression. Other mutations predicted to abolish the function of this gene have arisen recurrently in this gene, with multiple loss-of-expression alleles and premature stop codons segregating in natural populations. The frequency of these alleles varies greatly geographically, and in some southern African populations, natural selection has driven them near to fixation. We conclude that natural selection has favored the repeated loss of an important component of the immune system, suggesting that in some populations, a pleiotropic cost to lectin-24A expression outweighs the benefits of resistance.

Trans-regulatory changes underpin the evolution of the Drosophila immune response.

When an animal is infected, the expression of a large suite of genes is changed, resulting in an immune response that can defend the host. Despite much evidence that the sequence of proteins in the immune system can evolve rapidly, the evolution of gene expression is comparatively poorly understood. We therefore investigated the transcriptional response to parasitoid wasp infection in Drosophila simulans and D. sechellia. Although these species are closely related, there has been a large scale divergence in the expression of immune-responsive genes in their two main immune tissues, the fat body and hemocytes. Many genes, including those encoding molecules that directly kill pathogens, have cis regulatory changes, frequently resulting in large differences in their expression in the two species. However, these changes in cis regulation overwhelmingly affected gene expression in immune-challenged and uninfected animals alike. Divergence in the response to infection was controlled in trans. We argue that altering trans-regulatory factors, such as signalling pathways or immune modulators, may allow natural selection to alter the expression of large numbers of immune-responsive genes in a coordinated fashion.

Constitutive activation of cellular immunity underlies the evolution of resistance to infection in Drosophila.

Organisms rely on inducible and constitutive immune defences to combat infection. Constitutive immunity enables a rapid response to infection but may carry a cost for uninfected individuals, leading to the prediction that it will be favoured when infection rates are high. When we exposed populations of Drosophila melanogaster to intense parasitism by the parasitoid wasp Leptopilina boulardi, they evolved resistance by developing a more reactive cellular immune response. Using single-cell RNA sequencing, we found that immune-inducible genes had become constitutively upregulated. This was the result of resistant larvae differentiating precursors of specialized immune cells called lamellocytes that were previously only produced after infection. Therefore, populations evolved resistance by genetically hard-wiring the first steps of an induced immune response to become constitutive.

Independent effects on cellular and humoral immune responses underlie genotype-by-genotype interactions between Drosophila and parasitoids.

It is common to find abundant genetic variation in host resistance and parasite infectivity within populations, with the outcome of infection frequently depending on genotype-specific interactions. Underlying these effects are complex immune defenses that are under the control of both host and parasite genes. We have found extensive variation in Drosophila melanogaster's immune response against the parasitoid wasp Leptopilina boulardi. Some aspects of the immune response, such as phenoloxidase activity, are predominantly affected by the host genotype. Some, such as upregulation of the complement-like protein Tep1, are controlled by the parasite genotype. Others, like the differentiation of immune cells called lamellocytes, depend on the specific combination of host and parasite genotypes. These observations illustrate how the outcome of infection depends on independent genetic effects on different aspects of host immunity. As parasite-killing results from the concerted action of different components of the immune response, these observations provide a physiological mechanism to generate phenomena like epistasis and genotype-interactions that underlie models of coevolution.

Parallel and costly changes to cellular immunity underlie the evolution of parasitoid resistance in three Drosophila species.

A priority for biomedical research is to understand the causes of variation in susceptibility to infection. To investigate genetic variation in a model system, we used flies collected from single populations of three different species of Drosophila and artificially selected them for resistance to the parasitoid wasp Leptopilina boulardi, and found that survival rates increased 3 to 30 fold within 6 generations. Resistance in all three species involves a large increase in the number of the circulating hemocytes that kill parasitoids. However, the different species achieve this in different ways, with D. melanogaster moving sessile hemocytes into circulation while the other species simply produce more cells. Therefore, the convergent evolution of the immune phenotype has different developmental bases. These changes are costly, as resistant populations of all three species had greatly reduced larval survival. In all three species resistance is only costly when food is in short supply, and resistance was rapidly lost from D. melanogaster populations when food is restricted. Furthermore, evolving resistance to L. boulardi resulted in cross-resistance against other parasitoids. Therefore, whether a population evolves resistance will depend on ecological conditions including food availability and the presence of different parasite species.

Tetranychus urticae mites do not mount an induced immune response against bacteria.

The genome of the spider mite Tetranychus urticae, a herbivore, is missing important elements of the canonical Drosophila immune pathways necessary to fight bacterial infections. However, it is not known whether spider mites can mount an immune response and survive bacterial infection. In other chelicerates, bacterial infection elicits a response mediated by immune effectors leading to the survival of infected organisms. In T. urticae, infection by either Escherichia coli or Bacillus megaterium did not elicit a response as assessed through genome-wide transcriptomic analysis. In line with this, spider mites died within days even upon injection with low doses of bacteria that are non-pathogenic to Drosophila Moreover, bacterial populations grew exponentially inside the infected spider mites. By contrast, Sancassania berlesei, a litter-dwelling mite, controlled bacterial proliferation and resisted infections with both Gram-negative and Gram-positive bacteria lethal to T. urticae This differential mortality between mite species was absent when mites were infected with heat-killed bacteria. Also, we found that spider mites harbour in their gut 1000-fold less bacteria than S. berlesei We show that T. urticae has lost the capacity to mount an induced immune response against bacteria, in contrast to other mites and chelicerates but similarly to the phloem feeding aphid Acyrthosiphon pisum Hence, our results reinforce the putative evolutionary link between ecological conditions regarding exposure to bacteria and the architecture of the immune response.

Drosophila sessile hemocyte clusters are true hematopoietic tissues that regulate larval blood cell differentiation.

Virtually all species of coelomate animals contain blood cells that display a division of labor necessary for homeostasis. This functional partition depends upon the balance between proliferation and differentiation mostly accomplished in the hematopoietic organs. In Drosophila melanogaster, the lymph gland produces plasmatocytes and crystal cells that are not released until pupariation. Yet, throughout larval development, both hemocyte types increase in numbers. Mature plasmatocytes can proliferate but it is not known if crystal cell numbers increase by self-renewal or by de novo differentiation. We show that new crystal cells in third instar larvae originate through a Notch-dependent process of plasmatocyte transdifferentiation. This process occurs in the sessile clusters and is contingent upon the integrity of these structures. The existence of this hematopoietic tissue, relying on structure-dependent signaling events to promote blood homeostasis, creates a new paradigm for addressing outstanding questions in Drosophila hematopoiesis and establishing further parallels with vertebrate systems.

Steroid hormone signaling is essential to regulate innate immune cells and fight bacterial infection in Drosophila.

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.

Alternative splicing and gene duplication in the evolution of the FoxP gene subfamily.

The FoxP gene subfamily of transcription factors is defined by its characteristic 110 amino acid long DNA-binding forkhead domain and plays essential roles in vertebrate biology. Its four members, FoxP1-P4, have been extensively characterized functionally. FoxP1, FoxP2, and FoxP4 are involved in lung, heart, gut, and central nervous system (CNS) development. FoxP3 is necessary and sufficient for the specification of regulatory T cells (Tregs) of the adaptive immune system. In Drosophila melanogaster, in silico predictions identify one unique FoxP subfamily gene member (CG16899) with no described function. We characterized this gene and established that it generates by alternative splicing two isoforms that differ in the forkhead DNA-binding domain. In D. melanogaster, both isoforms are expressed in the embryonic CNS, but in hemocytes, only isoform A is expressed, hinting to a putative modulation through alternative splicing of FoxP1 function in immunity and/or other hemocyte-dependent processes. Furthermore, we show that in vertebrates, this novel alternative splicing pattern is conserved for FoxP1. In mice, this new FoxP1 isoform is expressed in brain, liver, heart, testes, thymus, and macrophages (equivalent in function to hemocytes). This alternative splicing pattern has arisen at the base of the Bilateria, probably through exon tandem duplication. Moreover, our phylogenetic analysis suggests that in vertebrates, FoxP1 is more related to the FoxP gene ancestral form and the other three paralogues, originated through serial duplications, which only retained one of the alternative exons. Also, the newly described isoform differs from the other in amino acids critical for DNA-binding specificity. The integrity of its fold is maintained, but the molecule has lost the direct hydrogen bonding to DNA bases leading to a putatively lower specificity and possibly affinity toward DNA. With the present comparative study, through the integration of experimental and in silico studies of the FoxP gene subfamily across the animal kingdom, we establish a new model for the FoxP gene in invertebrates and for the vertebrate FoxP1 paralogue. Furthermore, we present a scenario for the structural evolution of this gene class and reveal new previously unsuspected levels of regulation for FoxP1 in the vertebrate system.