Dynamic changes in Wolbachia infection over a single generation of Drosophila suzukii, across a wide range of resource availability.

Wolbachia bacteria are maternally inherited symbionts that commonly infect terrestrial arthropods. Many Wolbachia reach high frequencies in their hosts by manipulating their reproduction, for example by causing reproductive incompatibilities between infected male and uninfected female hosts. However, not all strains manipulate reproduction, and a key unresolved question is how these non-manipulative Wolbachia persist in their hosts, often at intermediate to high frequencies. One such strain, wSuz, infects the invasive fruit pest Drosophila suzukii, spotted-wing drosophila. Here, we tested the hypothesis that wSuz infection provides a competitive benefit when resources are limited. Over the course of one season, we established population cages with varying amounts of food in a semi-field setting and seeded them with a 50:50 mixture of flies with and without Wolbachia. We predicted that Wolbachia-infected individuals should have higher survival and faster development than their uninfected counterparts when there was little available food. We found that while food availability strongly impacted fly fitness, there was no difference in development times or survival between Wolbachia-infected and uninfected flies. Interestingly, however, Wolbachia infection frequencies changed dramatically, with infections either increasing or decreasing by as much as 30% in a single generation, suggesting the possibility of unidentified factors shaping Wolbachia infection over the course of the season.

A highly divergent Wolbachia with a tiny genome in an insect-parasitic tylenchid nematode.

Wolbachia symbionts are the most successful host-associated microbes on the planet, infecting arthropods and nematodes. Their role in nematodes is particularly enigmatic, with filarial nematode species either 100% infected and dependent on symbionts for reproduction and development, or not at all infected. We have discovered a highly divergent strain of Wolbachia in an insect-parasitic tylenchid nematode, Howardula sp., in a nematode clade that has not previously been known to harbour Wolbachia. While this nematode is 100% infected with Wolbachia, we did not detect it in related species. We sequenced the Howardula symbiont (wHow) genome and found that it is highly reduced, comprising only 550 kilobase pairs of DNA, approximately 35% smaller than the smallest Wolbachia nematode symbiont genomes. The wHow genome is a subset of all other Wolbachia genomes and has not acquired any new genetic information. While it has lost many genes, including genes involved in cell wall synthesis and cell division, it has retained the entire haem biosynthesis pathway, suggesting that haem supplementation is critical. wHow provides key insights into our understanding of what are the lower limits of Wolbachia cells, as well as the role of Wolbachia symbionts in the biology and convergent evolution of diverse parasitic nematodes.

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.

The adipokine NimrodB5 regulates peripheral hematopoiesis in Drosophila.

In animals, growth is regulated by the complex interplay between paracrine and endocrine signals. When food is scarce, tissues compete for nutrients, leading to critical resource allocation and prioritization. Little is known about how the immune system maturation is coordinated with the growth of other tissues. Here, we describe a signaling mechanism that regulates the number of hemocytes (blood cells) according to the nutritional state of the Drosophila larva. Specifically, we found that a secreted protein, NimB5, is produced in the fat body upon nutrient scarcity downstream of metabolic sensors and ecdysone signaling. NimB5 is then secreted and binds to hemocytes to down-regulate their proliferation and adhesion. Blocking this signaling loop results in conditional lethality when larvae are raised on a poor diet, due to excessive hemocyte numbers and insufficient energy storage. Similar regulatory mechanisms shaping the immune system in response to nutrient availability are likely to be widespread in animals.

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-κB 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.

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 NimC11 ;eater1 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.

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.

Drosophila innate immunity: regional and functional specialization of prophenoloxidases.

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.