Cellular Immunity of Drosophila willistoni Reveals Novel Complexity in Insect Anti-Parasitoid Defense.

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.

Evolution of insect innate immunity through domestication of bacterial toxins.

Toxin cargo genes are often horizontally transferred by phages between bacterial species and are known to play an important role in the evolution of bacterial pathogenesis. Here, we show how these same genes have been horizontally transferred from phage or bacteria to animals and have resulted in novel adaptations. We discovered that two widespread bacterial genes encoding toxins of animal cells, cytolethal distending toxin subunit B (cdtB) and apoptosis-inducing protein of 56 kDa (aip56), were captured by insect genomes through horizontal gene transfer from bacteria or phages. To study the function of these genes in insects, we focused on Drosophila ananassae as a model. In the D. ananassae subgroup species, cdtB and aip56 are present as singular (cdtB) or fused copies (cdtB::aip56) on the second chromosome. We found that cdtB and aip56 genes and encoded proteins were expressed by immune cells, some proteins were localized to the wasp embryo's serosa, and their expression increased following parasitoid wasp infection. Species of the ananassae subgroup are highly resistant to parasitoid wasps, and we observed that D. ananassae lines carrying null mutations in cdtB and aip56 toxin genes were more susceptible to parasitoids than the wild type. We conclude that toxin cargo genes were captured by these insects millions of years ago and integrated as novel modules into their innate immune system. These modules now represent components of a heretofore undescribed defense response and are important for resistance to parasitoid wasps. Phage or bacterially derived eukaryotic toxin genes serve as macromutations that can spur the instantaneous evolution of novelty in animals.

Distinctive features of Zaprionus indianus hemocyte differentiation and function revealed by transcriptomic analysis.

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.

Broad Ultrastructural and Transcriptomic Changes Underlie the Multinucleated Giant Hemocyte Mediated Innate Immune Response against Parasitoids.

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.

Cellular Immune Response Involving Multinucleated Giant Hemocytes with Two-Step Genome Amplification in the Drosophilid Zaprionus indianus.

Previously, a novel cell type, the multinucleated giant hemocyte (MGH) was identified in the ananassae subgroup of Drosophilidae. These cells share several features with mammalian multinucleated giant cells, a syncytium of macrophages formed during granulomatous inflammation. We were able to show that MGHs also differentiate in Zaprionus indianus, an invasive species belonging to the vittiger subgroup of the family, highly resistant to a large number of parasitoid wasp species. We have classified the MGHs of Z. indianusas giant hemocytes belonging to a class of cells which also include elongated blood cells carrying a single nucleus and anuclear structures. They are involved in encapsulating parasites, originate from the lymph gland, can develop by cell fusion, and generally carry many nuclei, while possessing an elaborated system of canals and sinuses, resulting in a spongiform appearance. Their nuclei are all transcriptionally active and show accretion of genetic material. Multinucleation and accumulation of the genetic material in the giant hemocytes represents a two-stage amplification of the genome, while their spongy ultrastructure substantially increases the contact surface with the extracellular space. These features may furnish the giant hemocytes with a considerable metabolic advantage, hence contributing to the mechanism of the effective immune response.