Project description:One function of plant lectins is to serve as defenses against herbivorous insects. The midgut is the critical site affected by dietary lectins such as wheat germ agglutinin (WGA). We observed marked cellular structural and gene expression changes in Drosophila melanogaster third-instar larval midguts from insects WGA-fed or starved. Dietary WGA caused shortening, branching, swelling, distortion and in some cases disintegration of the midgut microvilli (Mv). Starvation was accompanied by shortening of the Mv. Microarray analyses revealed that dietary WGA evolved differential expression of 62 transcripts; seven of which were also differentially expressed in starved insects. The differentially regulated gene cluster in WGA-fed larvae were associated with (i) cytoskeletal organization, (ii) immune responses, (iii) detoxification reactions, and (iv) energy metabolism. Four putative transcription factor binding motifs (TFBMs) were associated with the differentially-expressed genes. At least one of these putative TFBMs exhibited substantial similarity to MyoD, a TFBM associated with cellular structures in mammals. These results are in keeping with the hypothesis that WGA causes a starvation-like effect as well as structural changes of midgut cells of Drosophila third-instar larvae. Keywords: stress response
Project description:One function of plant lectins is to serve as defenses against herbivorous insects. The midgut is the critical site affected by dietary lectins such as wheat germ agglutinin (WGA). We observed marked cellular structural and gene expression changes in Drosophila melanogaster third-instar larval midguts from insects WGA-fed or starved. Dietary WGA caused shortening, branching, swelling, distortion and in some cases disintegration of the midgut microvilli (Mv). Starvation was accompanied by shortening of the Mv. Microarray analyses revealed that dietary WGA evolved differential expression of 62 transcripts; seven of which were also differentially expressed in starved insects. The differentially regulated gene cluster in WGA-fed larvae were associated with (i) cytoskeletal organization, (ii) immune responses, (iii) detoxification reactions, and (iv) energy metabolism. Four putative transcription factor binding motifs (TFBMs) were associated with the differentially-expressed genes. At least one of these putative TFBMs exhibited substantial similarity to MyoD, a TFBM associated with cellular structures in mammals. These results are in keeping with the hypothesis that WGA causes a starvation-like effect as well as structural changes of midgut cells of Drosophila third-instar larvae. Experiment Overall Design: Three treatments (control, WGA, and Starvation), and three replicates for each treatment. So there are total nine samples. no dye-swap.
Project description:The larval ovary is made up of multiple cell types including germ cells and somatic cells. The diversity of cell types and transcriptional regulation is not fully understood. To get single cell resolution of larval ovary regulation, we generated single-cell RNA expression profiles (scRNA-Seq) from late third instar larval ovaries of a reference Drosophila melanogaster genotype w[1118].
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.
Project description:The male larval gonad is a complex tissue made up of multiple cell types including germ cells and somatic cells. It is also a key developmental stage with the germline undergoing spermatogenesis. The diversity of cell types and transcriptional regulation of the developing gonad has only been broadly described. In order to get single cell resolution, we profiled late third instar larval testes of a reference Drosophila melanogaster genotype, w[1118] using single-cell RNA-Seq (scRNA-Seq). We identified and annotated 9 cell types including 4 stages of the germ cell lineage, and provide transcriptional profiles for each cell type.
Project description:Bowman-Birk Inhibitor (BBI) has both insecticidal and anti-cancerous properties. It has been hypothesized that dietary BBI slows insect growth by inhibiting the catalytic activity of digestive enzymes trypsins and chyomotrypsins, resulting in the midgut having reduced access to amino acids needed for growth. In mammals, BBI was hypothesized to influence cellular energy metabolism. Thus, we tested the hypothesis that dietary BBI also impacts energy-associated pathways in the midgut of Drosophila melanogaster. We investigated the impact of dietary BBI on the following parameters in the midguts of third-instar Drosophila larvae: (i) cellular metabolites, (ii) global transcriptome response, (iii) putative transcription factor binding sites (TFBSs) associated with the differentially expressed transcripts, and (iv) epithelial cellular structure. Dietary BBI caused: (i) a reduction of cellular DHAP, glucose, and succinate; and, (ii) increased Fructuse-6-phosphate; (ii) differential expression of genes associated with the glucose and fatty acid utilization; and, (iii) a shortening of midgut epithelial microvilli, a phenomenon previously associated with glucose starvation. Additionally, fifty seven percent of the putative TFBSs associated with the differentially expressed transcripts have previously been associated with glucose and insulin activities in mammalian studies. Collectively these results support the hypothesis that dietary BBI influences energy utilization in the Drosophila midgut. Keywords: stress response
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.