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Background: To combat infection, an immune system needs to be promptly activated but tightly controlled to avoid destruction of host tissues. IbinA and IbinB are related short peptides with robust expression upon microbial challenge in Drosophila melanogaster. Results: Ibin genes are ubiquitously present in flies of the Drosophila subgenus Sophophora, replacing the likely evolutionarily older, related gene, Mibin, which is found across a much wider range of cyclorrhaphan flies and is also upregulated following infection. We observed no direct bactericidal or bacteriostatic activity for IbinA or IbinB in vitro. Using single and double Ibin mutant Drosophila lines, we examined their roles in development and during microbial infections. IbinA is expressed in early pupae, and a lack of IbinA and IbinB leads to temperature-dependent formation of melanized tissue during metamorphosis, frequently around the trachea. IbinA and IbinB have distinct effects on susceptibility to microbial infection. For example, flies lacking IbinB had improved survival when challenged with Listeria monocytogenes, an intracellular pathogen, whereas a lack of IbinA alone had no effect. RNA sequencing following L. monocytogenes infection showed enhanced Toll target gene expression in flies lacking IbinB, suggesting that IbinB acts as a negative regulator of the Toll pathway. In contrast, IbinA mutants had decreased Toll target gene expression. Correspondingly, IbinB mutant flies had improved, and IbinA compromised survival in septic fungal infection, where the Toll pathway has a major role. Conclusions: Our study provides insight into the roles of IbinA and IbinB in regulation of the immune response in Drosophila.

Introduction: Species of the ananassae subgroup of Drosophilidae are highly resistant to parasitoid wasp infections. We have previously shown that the genes encoding cytolethal distending toxin B (CdtB) and the apoptosis inducing protein of 56 kDa (AIP56) were horizontally transferred to these fly species from prokaryotes and are now instrumental in the anti-parasitoid immune defense of Drosophila ananassae. Herewe describe a new family of genes, which encode proteins with hemolysin E domains, heretofore only identified in prokaryotes. Hemolysin E proteins are pore-forming toxins, important virulence factors of bacteria.

Methods: Bioinformatical, transcriptional, and protein expressional studies were used.

Results: The hemolysin E-like genes have a scattered distribution among the genomes of species belonging to several different monophyletic lineages in the family Drosophilidae. We detected structural homology with the bacterial Hemolysin E toxins and showed that the origin of the D. ananassae hemolysin E-like genes (hl1-38) is consistent with prokaryotic horizontal gene transfer. These genes encode humoral factors, secreted into the hemolymph by the fat body and hemocytes. Their expression is induced solely by parasitoid infection and the proteins bind to the developing parasitoids.

Conclusions: Hemolysin E-like proteins acquired by horizontal gene transfer and expressed by the primary immune organs may contribute to the elimination of parasitoids, as novel humoral factors in Drosophila innate immunity. 

Coevolution of hosts and their parasites has shaped heterogeneity of effector hemocyte types, providing immune defense reactions with variable effectiveness. In this work, we characterize hemocytes of Drosophila willistoni, a species that has evolved a cellular immune system with extensive variation and a high degree of plasticity. Monoclonal antibodies were raised and used in indirect immunofluorescence experiments to characterize hemocyte subpopulations, follow their functional features and differentiation. Pagocytosis and parasitization assays were used to determine the functional characteristics of hemocyte types. Samples were visualized using confocal and epifluorescence microscopy. We identified a new multinucleated giant hemocyte (MGH) type, which differentiates in the course of the cellular immune response to parasitoids. These cells differentiate in the circulation through nuclear division and cell fusion, and can also be derived from the central hematopoietic organ, the lymph gland. They have a binary function as they take up bacteria by phagocytosis and are involved in the encapsulation and elimination of the parasitoid. Here, we show that, in response to large foreign particles, such as parasitoids, MGHs differentiate, have a binary function and contribute to a highly effective cellular immune response, similar to the foreign body giant cells of vertebrates.

Background: Insects have specialized cell types that participate in the elimination of parasites, for instance, the lamellocytes of the broadly studied species Drosophila melanogaster. Other drosophilids, such as Drosophila ananassae and the invasive Zaprionus indianus, have multinucleated giant hemocytes, a syncytium of blood cells that participate in the encapsulation of the eggs or larvae of parasitoid wasps. These cells can be formed by the fusion of hemocytes in circulation or originate from the lymph gland. Their ultrastructure highly resembles that of the mammalian megakaryocytes.

Methods: Morphological, protein expressional, and functional features of blood cells were revealed using epifluorescence and confocal microscopy. The respective hemocyte subpopulations were identified using monoclonal antibodies in indirect immunofluorescence assays. Fluorescein isothiocyanate (FITC)-labeled Escherichia coli bacteria were used in phagocytosis tests. Gene expression analysis was performed following mRNA sequencing of blood cells.

Results: D. ananassae and Z. indianus encapsulate foreign particles with the involvement of multinucleated giant hemocytes and mount a highly efficient immune response against parasitoid wasps. Morphological, protein expressional, and functional assays of Z. indianus blood cells suggested that these cells could be derived from large plasmatocytes, a unique cell type developing specifically after parasitoid wasp infection. Transcriptomic analysis of blood cells, isolated from naïve and wasp-infected Z. indianus larvae, revealed several differentially expressed genes involved in signal transduction, cell movements, encapsulation of foreign targets, energy production, and melanization, suggesting their role in the anti-parasitoid response. A large number of genes that encode proteins associated with coagulation and wound healing, such as phenoloxidase activity factor-like proteins, fibrinogen-related proteins, lectins, and proteins involved in the differentiation and function of platelets, were constitutively expressed. The remarkable ultrastructural similarities between giant hemocytes and mammalian megakaryocytes, and presence of platelets, and giant cell-derived anucleated fragments at wound sites hint at the involvement of this cell subpopulation in wound healing processes, in addition to participation in the encapsulation reaction.

Conclusion: Our observations provide insights into the broad repertoire of blood cell functions required for efficient defense reactions to maintain the homeostasis of the organism. The analysis of the differentiation and function of multinucleated giant hemocytes gives an insight into the diversification of the immune mechanisms.

Multinucleated giant hemocytes (MGHs) represent a novel type of blood cell in insects that participate in a highly efficient immune response against parasitoid wasps involving isolation and killing of the parasite. Previously, we showed that circulating MGHs have high motility and the interaction with the parasitoid rapidly triggers encapsulation. However, structural and molecular mechanisms behind these processes remained elusive. Here, we used detailed ultrastructural analysis and live cell imaging of MGHs to study encapsulation in Drosophila ananassae after parasitoid wasp infection. We found dynamic structural changes, mainly driven by the formation of diverse vesicular systems and newly developed complex intracytoplasmic membrane structures, and abundant generation of giant cell exosomes in MGHs. In addition, we used RNA sequencing to study the transcriptomic profile of MGHs and activated plasmatocytes 72 h after infection, as well as the uninduced blood cells. This revealed that differentiation of MGHs was accompanied by broad changes in gene expression. Consistent with the observed structural changes, transcripts related to vesicular function, cytoskeletal organization, and adhesion were enriched in MGHs. In addition, several orphan genes encoding for hemolysin-like proteins, pore-forming toxins of prokaryotic origin, were expressed at high level, which may be important for parasitoid elimination. Our results reveal coordinated molecular and structural changes in the course of MGH differentiation and parasitoid encapsulation, providing a mechanistic model for a powerful innate immune response.

Hemocytes, similar to vertebrate blood cells, play important roles in insect development and immunity, but it is not well understood how they perform their tasks. New technology, in particular single-cell transcriptomic analysis in combination with Drosophila genetics, may now change this picture. This review aims to make sense of recently published data, focusing on Drosophila melanogaster and comparing to data from other drosophilids, the malaria mosquito, Anopheles gambiae, and the silkworm, Bombyx mori. Basically, the new data support the presence of a few major classes of hemocytes: (1) a highly heterogenous and plastic class of professional phagocytes with many functions, called plasmatocytes in Drosophila and granular cells in other insects. (2) A conserved class of cells that control melanin deposition around parasites and wounds, called crystal cells in D. melanogaster, and oenocytoids in other insects. (3) A new class of cells, the primocytes, so far only identified in D. melanogaster. They are related to cells of the so-called posterior signaling center of the larval hematopoietic organ, which controls the hematopoiesis of other hemocytes. (4) Different kinds of specialized cells, like the lamellocytes in D. melanogaster, for the encapsulation of parasites. These cells undergo rapid evolution, and the homology relationships between such cells in different insects are uncertain. Lists of genes expressed in the different hemocyte classes now provide a solid ground for further investigation of function.

Nora virus, a virus of Drosophila, encapsidates one of the largest single-stranded RNA virus genomes known. Its taxonomic affinity is uncertain as it has a picornavirus-like cassette of enzymes for virus replication, but the capsid structure was at the time for genome publication unknown. By solving the structure of the virus, and through sequence comparison, we clear up this taxonomic ambiguity in the invertebrate RNA virosphere. Despite the lack of detectable similarity in the amino acid sequences, the 2.7 angstrom resolution cryoEM map showed Nora virus to have T=1 symmetry with the characteristic capsid protein beta -barrels found in all the viruses in the Picornavirales order. Strikingly, alpha -helical bundles formed from the extended C-termini of capsid protein VP4B and VP4C protrude from the capsid surface. They are similar to signalling molecule folds and implicated in virus entry. Unlike other viruses of Picornavirales, no intra-pentamer stabilizing annulus was seen, instead the intra-pentamer stability comes from the interaction of VP4C and VP4B N-termini. Finally, intertwining of the N-termini of two-fold symmetry-related VP4A capsid proteins and RNA, provides inter-pentamer stability. Based on its distinct structural elements and the genetic distance to other picorna-like viruses we propose that Nora virus, and a small group of related viruses, should have its own family within the order Picornavirales.