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IntroductionChitinase-like proteins (CLPs) are associated with tissue-remodeling and inflammation but also with several disorders, including fibrosis, atherosclerosis, allergies, and cancer. However, CLP’s role in tumors is far from clear. MethodsHere, we utilize Drosophila melanogaster and molecular genetics to investigate the function of CLPs (imaginal disc growth factors; Idgf’s) in RasV12 dysplastic salivary glands. Results and discussionWe find one of the Idgf’s members, Idgf3, is transcriptionally induced in a JNK-dependent manner via a positive feedback loop mediated by reactive oxygen species (ROS). Moreover, Idgf3 accumulates in enlarged endosomal vesicles (EnVs) that promote tumor progression by disrupting cytoskeletal organization. The process is mediated via the downstream component, aSpectrin, which localizes to the EnVs. Our data provide new insight into CLP function in tumors and identifies specific targets for tumor control.

Endoparasitoid wasps are important natural enemies of many insect species and are major selective forces on the host immune system. Despite increased interest in insect antiparasitoid immunity, there is sparse information on the evolutionary dynamics of biological pathways and gene regulation involved in host immune defense outside Drosophila species. We de novo assembled transcriptomes from two beetle species and used time-course differential expression analysis to investigate gene expression differences in closely related species Galerucella pusilla and G. calmariensis that are, respectively, resistant and susceptible against parasitoid infection by Asecodes parviclava parasitoids. Approximately 271 million and 224 million paired-ended reads were assembled and filtered to form 52,563 and 59,781 transcripts for G. pusilla and G. calmariensis, respectively. In the whole-transcriptome level, an enrichment of functional categories related to energy production, biosynthetic process, and metabolic process was exhibited in both species. The main difference between species appears to be immune response and wound healing process mounted by G. pusilla larvae. Using reciprocal BLAST against the Drosophila melanogaster proteome, 120 and 121 immune-related genes were identified in G. pusilla and G. calmariensis, respectively. More immune genes were differentially expressed in G. pusilla than in G. calmariensis, in particular genes involved in signaling, hematopoiesis, and melanization. In contrast, only one gene was differentially expressed in G. calmariensis. Our study characterizes important genes and pathways involved in different immune functions after parasitoid infection and supports the role of signaling and hematopoiesis genes as key players in host immunity in Galerucella against parasitoid wasps.

Insects rely on their innate immune system to successfully mediate complex interactions with their internal microbiota, as well as the microbes present in the environment. Given the variation in microbes across habitats, the challenges to respond to them are likely to result in local adaptations in the immune system. Here we focus upon phagocytosis, a mechanism by which pathogens and foreign particles are engulfed in order to be contained, killed, and processed. We investigated the phenotypic and genetic variation related to phagocytosis in two allopatric populations of the butterfly Pieris napi. Populations were found to differ in their hemocyte composition and overall phagocytic capability, driven by the increased phagocytic propensity of each cell type. Yet, genes annotated to phagocytosis showed no large genomic signal of divergence. However, a gene set enrichment analysis on significantly divergent genes identified loci involved in glutamine metabolism, which recently have been linked to immune cell differentiation in mammals. Together these results suggest that heritable variation in phagocytic capacity arises via a quantitative trait architecture with variation in genes affecting the activation and/or differentiation of phagocytic cells, suggesting them as potential candidate genes underlying these phenotypic differences.

Many leukemia patients suffer from dysregulation of their immune system, making them more susceptible to infections and leading to general weakening (cachexia). Both adaptive and innate immunity are affected. The fruit fly Drosophila melanogaster has an innate immune system, including cells of the myeloid lineage (hemocytes). To study Drosophila immunity and physiology during leukemia, we established three models by driving expression of a dominant-active version of the Ras oncogene (RasV12) alone or combined with knockdowns of tumor suppressors in Drosophila hemocytes. Our results show that phagocytosis, hemocyte migration to wound sites, wound sealing, and survival upon bacterial infection of leukemic lines are similar to wild type. We find that in all leukemic models the two major immune pathways (Toll and Imd) are dysregulated. Toll–dependent signaling is activated to comparable extents as after wounding wild-type larvae, leading to a proinflammatory status. In contrast, Imd signaling is suppressed. Finally, we notice that adult tissue formation is blocked and degradation of cell masses during metamorphosis of leukemic lines, which is akin to the state of cancer-dependent cachexia. To further analyze the immune competence of leukemic lines, we used a natural infection model that involves insect-pathogenic nematodes. We identified two leukemic lines that were sensitive to nematode infections. Further characterization demonstrates that despite the absence of behavioral abnormalities at the larval stage, leukemic larvae show reduced locomotion in the presence of nematodes. Taken together, this work establishes new Drosophila models to study the physiological, immunological, and behavioral consequences of various forms of leukemia.

One of the key factors that determine the interaction between hosts and their parasites is the frequency of their interactions, which depends on the locomotory behavior of both parts. To address host behavior we used natural infections involving insect pathogenic nematodes and Drosophila melanogaster larvae as hosts. Using a modified version of a recently described method (FIMTrack) to assess several parameters in larger sets of animals, we initially detected specific differences in larval food searching when comparing Drosophila strains. These differences were further influenced by the presence of nematodes. Given a choice, Drosophila larvae clearly avoided nematodes irrespective of their genetic background. Our newly developed methods will be useful to test candidate genes and pathways involved in host/pathogen interactions in general and to assess specific parameters of their interaction.

Endoparasitoid wasps are important natural enemies of many insect species and are major selective forces on the host immune system. Despite increased interest in insect antiparasitoid immunity, there is sparse information on the evolutionary dynamics of biological pathways and gene regulation involved in host immune defense outside Drosophila species. We de novo assembled transcriptomes from two beetle species and used time-course differential expression analysis to investigate gene expression differences in closely related species Galerucella pusilla and G. calmariensis that are, respectively, resistant and susceptible against parasitoid infection by Asecodes paividava parasitoids. Approximately 271 million and 224 million paired-ended reads were assembled and filtered to form 52,563 and 59,781 transcripts for G. pusilla and G. calmariensis, respectively. In the whole-transcriptome level, an enrichment of functional categories related to energy production, biosynthetic process, and metabolic process was exhibited in both species. The main difference between species appears to be immune response and wound healing process mounted by G. pusilla larvae. Using reciprocal BLAST against the Drosophila melanogaster proteome, 120 and 121 immune-related genes were identified in G. pusilla and G. calmariensis, respectively. More immune genes were differentially expressed in G. pusilla than in G. calmariensis, in particular genes involved in signaling, hematopoiesis, and melanization. In contrast, only one gene was differentially expressed in G. calmariensis. Our study characterizes important genes and pathways involved in different immune functions after parasitoid infection and supports the role of signaling and hematopoiesis genes as key players in host immunity in Galerucella against parasitoid wasps.

Insects rely on their innate immune system to successfully mediate complex interactions with their internal microbiota, as well as the microbes present in the environment. Given the variation in microbes across habitats, the challenges to respond to them is likely to result in local adaptation in the immune system. Here we focus upon phagocytosis, a mechanism by which pathogens and foreign particles are engulfed in order to be contained, killed and processed for antigen presentation. We investigated the phenotypic and genetic variation related to phagocytosis, in two allopatric populations of the butterflyPieris napi. We found that the populations differ in their hemocyte composition, and overall phagocytic capability, driven by the increased phagocytic propensity of each cell type. However, no evidence for divergence in phagocytosis-related genes was observed, though an enrichment of genes involved in glutamine metabolism was found, which have recently been linked to immune cell differentiation in mammals.

Insect hemolymph coagulation: Kinetics of classically and non-classically secreted clotting factors Schmid et al., 2019. The linked article demonstrates the localization of two secretory proteins in Drosophila melanogaster, Prophenoloxidase (PPO2) and Transglutaminase-A (Tg) in hemocytes as well the clot with different tissue-specific drivers. Here we provide further data for the usefulness of the GFP-tagged version of the two crosslinking enzymes that are involved in clot hardening. The morphology of crystal cells is described using GFP-tagged PPO2 rather than with the use of antibodies in ex vivo hemolymph preparations. The use of the GFP-tagged proteins PPO2 and Tg is shown in additional contexts. Keywords: Insect immunity, Innate immunity, Non-classical secretion, Transglutaminase, Prophenoloxidase, Coagulation, Hemocytes

Term enrichment analysis of GO biological process terms for comparisons among four genome-wide transcription studies of D. melanogaster. 3wD, three weeks diapause (whole animals) our study; 1wD, abdomens sampled after one week diapause [140]; cold intolerance [141] and starvation [143]. Comparison based on genes in the Venn diagram in Fig. 10b. Term analysis of Groups A – H are shown separately. (PDF 1267 kb)

GSEA of KEGG pathway analysis. (A) The set of KEGG pathways enriched for upregulated genes encompass mostly carbohydrate metabolic pathways, drug metabolism and circadian rhythm pathway. (B) The set of KEGG pathways enriched for downregulated genes covers mostly RNA and DNA metabolism and protein degradation. Genes with |log2FC| ≥ 0.04 and q ≤ 0.05 were considered significantly regulated. Pathways highlighted in italics were defined only on the basis of experiments performed in mammalian systems. (PDF 45 kb)

1. Host-parasitoid systems are characterized by a continuous development of new defence strategies in hosts and counter-defence mechanisms in parasitoids. This co-evolutionary arms race makes host-parasitoid systems excellent for understanding trade-offs in host use caused by evolutionary changes in host immune responses and parasitoid virulence. However, knowledge obtained from natural host-parasitoid systems on such trade-offs is still limited. 2. In this study, the aim was to examine trade-offs in parasitoid virulence in Asecodes parviclava (Hymenoptera: Eulophidae) when attacking three closely related beetles: Galerucella pusilla, Galerucella calmariensis and Galerucella tenella (Coleoptera: Chrysomelidae). A second aim was to examine whether geographic variation in parasitoid infectivity or host immune response could explain differences in parasitism rate between northern and southern sites. 3. More specifically, we wanted to examine whether the capacity to infect host larvae differed depending on the previous host species of the parasitoids and if such differences were connected to differences in the induction of host immune systems. This was achieved by combining controlled parasitism experiments with cytological studies of infected larvae. 4. Our results reveal that parasitism success in A. parviclava differs both depending on previous and current host species, with a higher virulence when attacking larvae of the same species as the previous host. Virulence was in general high for parasitoids from G. pusilla and low for parasitoids from G. calmariensis. At the same time, G. pusilla larvae had the strongest immune response and G. calmariensis the weakest. These observations were linked to changes in the larval hemocyte composition, showing changes in cell types important for the encapsulation process in individuals infected by more or less virulent parasitoids. 5. These findings suggest ongoing evolution in parasitoid virulence and host immune response, making the system a strong candidate for further studies on host race formation and speciation.

SPIA of KEGG pathways. Significantly inhibited pathways are highlighted in blue and significantly activated ones in orange. Genes with |log2FC| ≥ 0.1 and q ≤ 0.05 were considered significantly regulated. Pathways highlighted in italics were defined only on the basis of experiments performed in mammalian systems. (PDF 42 kb)

Results of overrepresentation test of GO biological process terms for comparisons between our genome-wide transcription study of D. melanogaster dormancy and aging studies. 3wD, three weeks diapause (whole animals) our study; Aging, aging response genes [136] and candidate lifespan extending genes [135]. Comparison based on genes in the Venn diagram in Fig. 10a. Term enrichment analysis of Groups A – D are shown separately. (PDF 405 kb)

SPIA of KEGG pathways. Significantly inhibited pathways are highlighted in blue and significantly activated ones in orange. Genes with |log2FC| ≥ 0.1 and q ≤ 0.05 were considered significantly regulated. Pathways highlighted in italics were defined only on the basis of experiments performed in mammalian systems. (PDF 42 kb)

GSEA of KEGG pathway analysis. (A) The set of KEGG pathways enriched for upregulated genes encompass mostly carbohydrate metabolic pathways, drug metabolism and circadian rhythm pathway. (B) The set of KEGG pathways enriched for downregulated genes covers mostly RNA and DNA metabolism and protein degradation. Genes with |log2FC| ≥ 0.04 and q ≤ 0.05 were considered significantly regulated. Pathways highlighted in italics were defined only on the basis of experiments performed in mammalian systems. (PDF 45 kb)

Primers used for quantitative real-time PCR. (PDF 55 kb)

Detailed data on genes in seven signal pathways. Description of genes shown in Figs. 4, 5, 7, 9 and Additional file 8: Figure S4 and discussed in the corresponding sections of the paper. References are also given for these genes. Note that the genes are listed as they are shown in the figures (from top to bottom in the pathways). (PDF 328 kb)

Detailed data on genes in seven signal pathways. Description of genes shown in Figs. 4, 5, 7, 9 and Additional file 8: Figure S4 and discussed in the corresponding sections of the paper. References are also given for these genes. Note that the genes are listed as they are shown in the figures (from top to bottom in the pathways). (PDF 328 kb)

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are expressed in spatial relation to each other in situ. Multi-target chromogenic whole-mount in situ hybridization (MC-WISH) greatly facilitates the instant comparison of gene expression patterns, as it allows distinctive visualization of different mRNA species in contrasting colors in the same sample specimen. This provides the possibility to relate gene expression domains topographically to each other with high accuracy and to define unique and overlapping expression sites. In the presented protocol, we describe a MC-WISH procedure for comparing mRNA expression patterns of different genes in Drosophila embryos. Up to three RNA probes, each specific for another gene and labeled by a different hapten, are simultaneously hybridized to the embryo samples and subsequently detected by alkaline phosphatase-based colorimetric immunohistochemistry. The described procedure is detailed here for Drosophila, but works equally well with zebrafish embryos.

Chitinase-like proteins (CLPs) are associated with tissue-remodelling and inflammation but also with several disorders, including fibrosis, atherosclerosis, allergies, and cancer. However, the CLP's role in tumors is far from clear. Here, we utilize Drosophila melanogaster to investigate the function of CLPs (imaginal disc growth factors; Idgf's) in RasV12 dysplastic salivary glands. We find one of the Idgf's members, Idgf3, is transcriptionally induced tissue and cell-autonomously and in a non-canonical JNK-dependent manner via a positive feedback loop mediated by reactive oxygen species (ROS). Moreover, Idgf3 accumulates in enlarged endosomal vesicles (EVs) that promote tumor progression by disrupting cytoskeletal organization, independent of Rab5 and Rab11. The process is mediated via Rac1 and the downstream component, αSpectrin, localized to the EVs. Our data provide new insight into tissue-autonomous CLP function in tumors and identifies specific targets for tumor control.