The melanization reaction in Drosophila: More than black or white

The melanization reaction is a rapid and important immune mechanism in arthropods. It results in the production of melanin at the site of injury and around invading microbes. The enzymes responsible for melanogenesis are phenoloxidases (PO), which catalyze the oxidation of phenols to quinones, which then polymerize into melanin. A by-product of melanogenesis are cytotoxic compounds that can pose a threat to the host organism itself due to their unspecific effects. Melanogenesis is therefore tightly regulated: POs are produced in an inactive form as prophenoloxidases (PPOs), which get activated by the sequential cleavage of an extracellular serine protease (SP) cascade. The aims of this PhD thesis were to better understand the melanization reaction in Drosophila melanogaster, at both the effector and the regulation levels. D. melanogaster has three PPO genes. We generated for the first time a mutant for the third PPO gene, PPO3, and analyzed its function. We demonstrated that PPO3 has an important role in the melanotic encapsulation reaction, a defense mechanism against parasitization. Additionally, we extended our knowledge about the other PPOs, PPO1 and PPO2. We confirmed their role in the defense against septic infections and ascribed a new role for PPO2 in the melanotic encapsulation. The use of single or combined mutations allowed us to show that each PPO mutant has a specific phenotype, and that knocking out two of three genes is required to abolish a particular function completely. Thus, Drosophila PPOs have partially overlapping functions to optimize melanization. Finally, we demonstrated that PPO3 is the result of a gene duplication of PPO2, restricted to a subgroup of Drosophila, and likely evolved as an additional defense mechanism in the cellular encapsulation process, probably due to the evolutionary pressure from parasitoid wasps. In the second part of this thesis, we re-addressed the roles and functions of three SPs involved in the melanization process. We developed a novel screening method for defects in melanization by infecting flies with a low-dose of S. aureus. We found that only one of the three SPs, Sp7, is involved in survival upon septic infections. Additionally, we demonstrated that the melanization reaction resulting in the clearance of systemic infections is regulated by extracellular components of the Toll pathway. While a connection between the SPs regulating the melanization and the Toll pathway was found in other insects, our study provides the first demonstration in D. melanogaster. We also present evidence of a disconnect between the melanin production at wound sites and the melanization reaction resulting in the clearance of infections, indicating a role of cytotoxic compounds in the killing of microbes. Finally, we ascribed a new role to Hayan, another SP implicated in melanization. Hayan is dispensable for the clearance of septic infections, but is important for wound melanization. We also demonstrated that Hayan plays an important role in the activation of the Toll pathway. Hayan, together with the SP Persephone, is necessary to activate Toll after infection. We propose that both Hayan and Persephone are the result of a recent gene duplication event. This can explain why they still have overlapping functions, but these genes also show signs of early sub-functionalization. Collectively, our work provides important insights on both melanization and the activation of the Toll pathway

A rapid method to monitor parasitoid infestations in Drosophila suzukii populations

Parasitoide Wespen tragen zur Kon­trol­le von Insektenpopulationen bei und sind daher ein wichtiger Baustein zur biologischen Bekämpfung invasiver Schädlinge. Während der Ausbreitung von invasiven Schädlingen in neue Gebiete folgen ihnen häufig Parasitoide aus ihren Heimatregionen nach. Die Überwachung der einheimischen und der adventiven Parasitoidpopulationen und die Auswertung ihres Erfolgs bei der Reduzierung von Schädlingspopulationen im Freiland ist aufwändig, und beruht meist auf langwierigen Parasitierungs- und Schlupfversuchen. Wir haben eine schnelle und kostengünstige Methode zum Nachweis von Parasitoiden bei der invasiven Kirschessigfliege, Drosophila suzukii, entwickelt. Sie basiert auf dem Amplifizieren der Hymenoptera-spezifischen 28S rRNA Sequenz von DNA oder RNA aus Fliegenpuppen, welche den Nachweis von Parasitoiden bis auf Artniveau ermöglicht.Parasitoid wasps are efficient natural enemies of other insects. They are in focus as an important biological pest management element to control invasive pest species. While spreading into new areas these invasive pests are often followed by their natural parasitoids from their home regions. The monitoring of native and adventive parasitoids, and their success in controlling the pest populations in the field, is cumbersome and relies mainly on hatching experiments. Here, we present a rapid method to detect parasitoid infestations in the invasive agricultural pest Drosophila suzukii. We specifically amplified a hymenopteran 28S rRNA sequence from DNA or RNA extracted from fly pupae. This allowed the determination of parasitoid infestations at the species level

Drosophila innate immunity: regional and functional specialization of prophenoloxidases

The diversification of immune systems during evolution involves the expansion of particular gene families in given phyla. A better understanding of the metazoan immune system requires an analysis of the logic underlying such immune gene amplification. This analysis is now within reach due to the ease with which we can generate multiple mutations in an organism. In this paper, we analyze the contribution of the three Drosophila prophenoloxidases (PPOs) to host defense by generating single, double and triple mutants. PPOs are enzymes that catalyze the production of melanin at the site of infection and around parasites. They are the rate-limiting enzymes that contribute to the melanization reaction, a major immune mechanism of arthropods. The number of PPO-encoding genes is variable among insects, ranging from one in the bee to ten in the mosquito

Remote Control of Intestinal Stem Cell Activity by Haemocytes in Drosophila

The JAK/STAT pathway is a key signaling pathway in the regulation of development and immunity in metazoans. In contrast to the multiple combinatorial JAK/STAT pathways in mammals, only one canonical JAK/STAT pathway exists in Drosophila. It is activated by three secreted proteins of the Unpaired family (Upd): Upd1, Upd2 and Upd3. Although many studies have established a link between JAK/STAT activation and tissue damage, the mode of activation and the precise function of this pathway in the Drosophila systemic immune response remain unclear. In this study, we used mutations in upd2 and upd3 to investigate the role of the JAK/STAT pathway in the systemic immune response. Our study shows that haemocytes express the three upd genes and that injury markedly induces the expression of upd3 by the JNK pathway in haemocytes, which in turn activates the JAK/STAT pathway in the fat body and the gut. Surprisingly, release of Upd3 from haemocytes upon injury can remotely stimulate stem cell proliferation and the expression of Drosomycin-like genes in the intestine. Our results also suggest that a certain level of intestinal epithelium renewal is required for optimal survival to septic injury. While haemocyte-derived Upd promotes intestinal stem cell activation and survival upon septic injury, haemocytes are dispensable for epithelium renewal upon oral bacterial infection. Our study also indicates that intestinal epithelium renewal is sensitive to insults from both the lumen and the haemocoel. It also reveals that release of Upds by haemocytes coordinates the wound-healing program in multiple tissues, including the gut, an organ whose integrity is critical to fly survival

Comparative RNA-Seq analyses of Drosophila plasmatocytes reveal gene specific signatures in response to clean injury and septic injury.

Drosophila melanogaster's blood cells (hemocytes) play essential roles in wound healing and are involved in clearing microbial infections. Here, we report the transcriptional changes of larval plasmatocytes after clean injury or infection with the Gram-negative bacterium Escherichia coli or the Gram-positive bacterium Staphylococcus aureus compared to hemocytes recovered from unchallenged larvae via RNA-Sequencing. This study reveals 676 differentially expressed genes (DEGs) in hemocytes from clean injury samples compared to unchallenged samples, and 235 and 184 DEGs in E. coli and S. aureus samples respectively compared to clean injury samples. The clean injury samples showed enriched DEGs for immunity, clotting, cytoskeleton, cell migration, hemocyte differentiation, and indicated a metabolic reprogramming to aerobic glycolysis, a well-defined metabolic adaptation observed in mammalian macrophages. Microbial infections trigger significant transcription of immune genes, with significant differences between the E. coli and S. aureus samples suggesting that hemocytes have the ability to engage various programs upon infection. Collectively, our data bring new insights on Drosophila hemocyte function and open the route to post-genomic functional analysis of the cellular immune response

More Than Black or White: Melanization and Toll Share Regulatory Serine Proteases in Drosophila

The melanization response is an important defense mechanism in arthropods. This reaction is mediated by phenoloxidases (POs), which are activated by complex extracellular serine protease (SP) cascades. Here, we investigate the role of SPs in the melanization response using compound mutants in D. melanogaster and discover phenotypes previously concealed in single-mutant analyses. We find that two SPs, Hayan and Sp7, activate the melanization response in different manners: Hayan is required for blackening wound sites, whereas Sp7 regulates an alternate melanization reaction responsible for the clearance of Staphylococcus aureus. We present evidence that Sp7 is regulated by SPs activating the Toll NF-kappa B pathway, namely ModSP and Grass. Additionally, we reveal a role for the combined action of Hayan and Psh in propagating Toll signaling downstream of pattern recognition receptors activating either Toll signaling or the melanization response

Drosophila innate immunity: regional and functional specialization of prophenoloxidases

Abstract Background The diversification of immune systems during evolution involves the expansion of particular gene families in given phyla. A better understanding of the metazoan immune system requires an analysis of the logic underlying such immune gene amplification. This analysis is now within reach due to the ease with which we can generate multiple mutations in an organism. In this paper, we analyze the contribution of the three Drosophila prophenoloxidases (PPOs) to host defense by generating single, double and triple mutants. PPOs are enzymes that catalyze the production of melanin at the site of infection and around parasites. They are the rate-limiting enzymes that contribute to the melanization reaction, a major immune mechanism of arthropods. The number of PPO-encoding genes is variable among insects, ranging from one in the bee to ten in the mosquito. Results By analyzing mutations alone and in combination, we ascribe a specific function to each of the three PPOs of Drosophila. Our study confirms that two PPOs produced by crystal cells, PPO1 and PPO2, contribute to the bulk of melanization in the hemolymph, upon septic or clean injury. In contrast, PPO3, a PPO restricted to the D. melanogaster group, is expressed in lamellocytes and contributes to melanization during the encapsulation process. Interestingly, another overlapping set of PPOs, PPO2 and PPO3, achieve melanization of the capsule upon parasitoid wasp infection. Conclusions The use of single or combined mutations allowed us to show that each PPO mutant has a specific phenotype, and that knocking out two of three genes is required to abolish fully a particular function. Thus, Drosophila PPOs have partially overlapping functions to optimize melanization in at least two conditions: following injury or during encapsulation. Since PPO3 is restricted to the D. melanogaster group, this suggests that production of PPO by lamellocytes emerged as a recent defense mechanism against parasitoid wasps. We conclude that differences in spatial localization, immediate or late availability, and mode of activation underlie the functional diversification of the three Drosophila PPOs, with each of them having non-redundant but overlapping functions

Two Nimrod receptors, NimC1 and Eater, synergistically contribute to bacterial phagocytosis in Drosophila melanogaster

Eater and NimC1 are transmembrane receptors of the Drosophila Nimrod family, specifically expressed in haemocytes, the insect blood cells. Previous ex vivo and in vivoRNAi studies have pointed to their role in the phagocytosis of bacteria. Here, we have created a novel NimC1 null mutant to re-evaluate the role of NimC1, alone or in combination with Eater, in the cellular immune response. We show that NimC1 functions as an adhesion molecule ex vivo, but in contrast to Eater it is not required for haemocyte sessility in vivo. Ex vivo phagocytosis assays and electron microscopy experiments confirmed that Eater is the main phagocytic receptor for Gram-positive, but not Gram-negative bacteria, and contributes to microbe tethering to haemocytes. Surprisingly, NimC1 deletion did not impair phagocytosis of bacteria, nor their adhesion to the haemocytes. However, phagocytosis of both types of bacteria was almost abolished in NimC1(1);eater(1) haemocytes. This indicates that both receptors contribute synergistically to the phagocytosis of bacteria, but that Eater can bypass the requirement for NimC1. Finally, we uncovered that NimC1, but not Eater, is essential for uptake of latex beads and zymosan particles. We conclude that Eater and NimC1 are the two main receptors for phagocytosis of bacteria in Drosophila, and that each receptor likely plays distinct roles in microbial uptake

Additional file 2: Figure S2. of Drosophila innate immunity: regional and functional specialization of prophenoloxidases

Comparative genomics of the PPO3 locus. Shown are the best pairwise genome alignments for 22 Drosophila species versus D. melanogaster based on the UCSC genome browser net tracks. Species in the D. melanogaster group plus D. eugracilis have only one net that aligns contiguously the entire PPO3 microsyntenic region. Conversely, other species (besides D. takahashii) have a gap in the alignment at PPO3 that is filled by a secondary net that arises from alignment to paralogous sequences elsewhere in the genome. D. takahashii has only a single alignment net in the PPO3 region; however, this net is fragmented precisely in the PPO3 gene, consistent with the possible presence of non-canonical PPO3-like gene sequences in this species (PDF 1061 kb