Project description:The object of the microarray data is to find several genes effected by fungal infection. In order to establish the gene expressino levels in response to fungal infection in hemocytes, B.bassiana were injected into the posterior end of third instar larvae using a tungsten needle and incubated for 1h at 25 celcius degree. To compare with non-infected hemocytes, total RNA from infected hemocytes were labeled by Cy5, total RNA from non-infected hemocytes were labeled by Cy3, and hybridized to chip.
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:Drosophila adult blood cells originate from larval peripheral hemocytes (derived from the embryonic waves of hematopoiesis) and from larval lymph gland hemocytes. To gain insight into the expression repertoire of Drosophila blood cells, we established the gene expression profiles the adult blood cells and their ascendants.
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.