Project description:Upon pathogenic infection, drosophila larval host mounts an immune response. Parasitic wasps inject venom that contain virulence factors during oviposition, which can elicit host immune response, and in some cases, suppress host immune responses altogether. Several microarray experiments have been performed on different classes of parasitic wasps. We wanted to compare how Ganaspis xanthopoda-infected hosts respond compared to other classes of parasitic wasps. Third instar y w larvae from a 2-day egglay were infected with G. xanthopoda for three and six hours, respectively, by introducing waps in petri-dish containing larvae. Controls were handled side-by-side without introducing wasps. Host larvae were immediately dissected, infection confirmed by presence of wasp egg, and frozen in liquid nitrogen and ground in Trizol. RNA was isolated and checked by agarose gel-electrophoresis. Samples were then sent to the Microarray Core Facility at Weill Cornell Medical College.
Project description:Upon pathogenic infection, drosophila larval host mounts an immune response. Parasitic wasps inject venom that contain virulence factors during oviposition, which can elicit host immune response, and in some cases, suppress host immune responses altogether. Several microarray experiments have been performed on different classes of parasitic wasps. We wanted to compare how Ganaspis xanthopoda-infected hosts respond compared to other classes of parasitic wasps.
Project description:In Drosophila melanogaster larval hemolymph, under normal conditions, plasmatocytes and crystal cells represent respectively ~95% and ~5% of hemocytes, while lamellocytes, the third larval cell type, are absent since they are only induced after parasitoid wasp oviposition, their role being the encapsulation-melanization response to eliminate the wasp egg. However, even after induction lamellocytes number remains low, making difficult biochemical studies. Here using the D. melanogaster hopTum-l mutant that constitutively produces a high number of hemocytes, we set up a method to purify lamellocytes and analyzed their major proteins by 2D gel electrophoresis and their biotinylated plasma membrane surface proteins by 1D SDS-PAGE after affinity purification. Mass spectrometry allowed to identify 430 proteins from the 2D spots and 344 from affinity purified proteins, totalizing 639 unique proteins. Known lamellocyte markers such as PPO3 and the integrin myospheroid are among the major proteins and affinity purification led to the detection of other integrins and a large array of integrins associated proteins involved in cell-cell junction formation and function. Overall newly identified proteins indicated that these cells are highly adapted to the encapsulation process but may have also several different physiological functions. This study provides the basis for new lamellocyte studies in vivo and in vitro, and develop markers to search whether different populations coexist, establish their origins and decipher their respective roles in drosophila physiology and immunity.
Project description:Although host-parasitoid interactions are becoming well characterized at the organismal and cellular levels, much remains to be understood of the molecular bases for the host immune response and the parasitoids’ ability to defeat this immune response. Leptopilina boulardi and L. heterotoma, two closely related, highly infectious natural parasitoids of Drosophila melanogaster, appear to use very different infection strategies at the cellular level. Here, we further characterize cellular level differences in the infection characteristics of these two wasp species using newly derived, virulent inbred strains, and then use whole genome microarrays to compare the transcriptional response of Drosophila to each. While flies attacked by the melanogaster group specialist Leptopilina boulardi (strain Lb17) up-regulate numerous genes encoding proteolytic enzymes, components of the Toll and JAK/STAT pathways, and the melanization cascade as part of a combined cellular and humoral innate immune response, flies attacked by the generalist L. heterotoma (strain Lh14) do not appear to initiate an immune transcriptional response at the time points post-infection we assayed, perhaps due to the rapid venom-mediated lysis of host hemocytes (blood cells). Thus, the specialist parasitoid appears to invoke a full-blown immune response in the host, but suppresses and/or evades downstream components of this response. Given that activation of the host immune response likely depletes the energetic resources of the host, the specialist’s infection strategy seems relatively disadvantageous. However, we uncover the mechanism for one potentially important fitness tradeoff of the generalist’s highly immune suppressive infection strategy. Keywords: Time series of transcriptional responses against pathogens.
Project description:Drosophila melanogaster is a validated eukaryotic model for immunity-concerned studies in the post-genomic era. In the present study we performed oral experimental infection of D. melanogaster with Pseudomonas aeruginosa (strain ATCC27853). By using a whole genome microarray approach, we intended to identify significant alterations in the expression profile of relevant genes amenable to qualify as new models for the investigation of specific host-parasite interactions.
Project description:The innate immune response of insects relies on several humoral and cellular mechanisms that require the activation of circulating proteases in the hemolymph to be functional. Here, we analyzed the gelatinase and caseinase activities of Drosophila larval hemolymph under normal and pathogenic conditions (bacterial lipopolysaccharides or endoparasitoid Leptopilina boulardi) using in gel zymography. Gelatinase activity was more intense than caseinase activity and qualitative and quantitative variations were observed between D. melanogaster strains and Drosophila species. Mass spectrometry identified a large number of serine proteases in gel bands equivalent to the major gelatinase and caseinase bands and of these, the most abundant and redundant were Tequila and members of the Jonah and Trypsin protease families. However, hemolymph from Tequila null mutant larvae showed no obvious changes in zymographic bands. Nor did we observe any significant changes in hemolymph gelatinases activity 24 h after injection of bacterial lipopolysaccharides or after oviposition by endoparasitoid wasps. These data confirmed that many serine proteases are present in Drosophila larval hemolymph but those with gelatinase and caseinase activity may not change drastically during the immune response.