Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Microbiota-induced changes in Drosophila melanogaster host gene expression

ABSTRACT: To understand the effects of the microbiome of Drosophila melanogaster on host gene expression, we compared the transcriptome of guts from conventionally reared flies to their axenically (germ-free)-reared counterparts. Our analysis used dissected intestines from 4-7 day-old adult females and included two wild-type fly lines, OregonR and CantonS, as well as an immune-deficient line, RelishE20. With one of the wild-type lines, CantonS, we also looked at the impact of microbiome on the transcriptional profile of dissected intestines from aged cohorts (35-40 day-old females) and young (4-7 day-old) non-gut tissues (all tissues remaining from samples dissected for the analysis of guts.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Nichole Broderick

PROVIDER: E-MTAB-2447 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Innate immune responses rely on expression of potent effector molecules, such as antimicrobial peptides, which have the capability to kill invading microorganisms. The presence and recognition of microbial components triggers several signaling pathways, such as the Toll and IMD pathways, which in turn activate NF-kB/Rel transcription factors to induce transcription of a large number of immune system genes. Not much is known how these genes are kept silent in healthy flies in the presence of commensal microorganisms, and how the expression of immune defense genes is turned off. We found that several immune defense genes are constitutively active in nub[1] mutants, indicating that the POU domain transcription factor Pdm1/Nubbin may act as a repressor of immune gene expression. We used microarrays to analyze the global changes in gene expression in nub[1] mutants compared to wild type. To analyze changes in gene expression in the gut distinctly from other immune-responsive tissues, such as fat body and hemocytes, flies were dissected and the gut was analyzed separately from the rest of the fly (carcass). A large number of genes were differentially expressed in nub[1] mutants compared to wild type. The differentially expressed transcripts are dominated by genes involved in Immune system processes and in Metabolism/Catabolism processes. The gut samples were dominated by genes involved in biological processes linked to Metabolism/Catabolism, but also here, genes involved in Response processes were enriched among the differentially expressed genes. The following Drosophila melanogaster strains were used: OregonR was used as wild type control and nub[1] b[1] pr[1] was used as the nub[1] mutant. Flies were dissected so that gut tissue was analyzed separately (gut) from the rest of the fly, minus head and gut (carcass). In total, 12 samples consisting of four different sample types: wt gut, wt carcass, nub[1] gut and nub[1] carcass, and three samples of each type.

Project description:Summary: Multi-toxins Bt-crops carrying insecticidal toxins with similar host spectrum and different mode of action e.g. Cry and Vip, are expected to improve resistance management in target pests. Control failure has been informed for Cry toxins but not for Vip3A, of which no mechanism of resistance has yet been identified. Here we applied HT-SuperSAGE to analyze the transcriptome profiling in the midgut tissue of a tobacco budworm Heliothis virescens (F.) strain laboratory-selected for Vip3A resistance. A total of 1324252 26-bp tags were sequenced representing 17751 unique transcripts (UniTags) from genetically similar Vip3A-resistant (Vip-Sel) strain and susceptible control (Vip-Unsel) strain. Differential expression was found significant (≥ 2.5-fold or ≤ 0.4) for 1845 unigenes that constitute 10.4% of the total number of UniTags, where 277 represented overexpressed (OE) and 1568 underexpressed (UE) genes in Vip-Sel compared to Vip-Unsel. BLASTN searches mapped 1141 of these UniTags to H. virescens EST sequences, of which, 816 (143 OE and 673 UE) were unambiguously annotated to proteins in NCBI non-redundant protein databases. Gene ontology revealed Vip3A adaptation induced major constitutive transcriptional differences in serine-proteases (SP)-mediated proteolysis, ribosome biogenesis and metabolic processes. Several unigenes homologous to a particular member of the REsponse to PAThogen (REPAT) family were found to be predominantly OE. Since UniTags related to SP and ribosomal proteins (RP) were the most represented in the libraries, they were further analyzed in details. Interestingly, UniTags related to the putative Vip3A-binding protein RpS2 were underexpressed, while, the tumorigenesis suppressor RpL37 accounted for the 35% of total overexpressed RP. A subset of unigenes was chosen to confirm the HT-SuperSAGE data by qRT-PCR. The present study is the first providing a lepidopteran gut transcriptome associated with Vip3A resistance and a foundation for future attempts to elucidate the resistance mechanism. Methods: Midgut transcriptional profiles of third-instar larvae from genetically similar Vip3A-resistant (Vip-Sel) and susceptible control (Vip-Unsel) strains were generated by HT-SuperSAGE. RNA pools of Vip-Sel and Vip-Unsel samples (five RNA preparations each) were prepared and used for the construction of SuperSAGE libraries HvR_GCCT and HvS_GCAC, respectively, according to the procedure described by [Matsumura et al., 2010]. Purified PCR products were mixed and applied to Illumina Genome Analyzer II sequencing with GEX (DpnII) primer in the sequencing reactions as recommended by the manufacturer. Sorting of sequence reads based on index sequences and the subsequent extraction of sequence tags from reads was conducted using a script written in Perl. Fold-change for each tag was calculated as in [Gilardoni et al., 2010]. qRT–PCR validation was performed using TaqMan and SYBR Green assays.

Project description:Tsetse flies (Glossina spp.) are major vectors of African trypanosomes, causing either Human or Animal African Trypanosomiasis (HAT or AAT). Several approaches are developed to control the disease among which the anti-vector Sterile Insect Technique. Another approach in the frame of anti-vector strategies could consist in controlling the fly’s vector competence which needs identifying factors (genes, proteins, biological pathways, …) involved in this process. The present work aims to verify whether protein candidates identified under experimental controlled conditions on insectary-reared tsetse flies have their counterpart in field-collected flies. Glossina palpalis palpalis flies naturally infected with Trypanosoma congolense were sampled in two HAT/AAT foci in Southern Cameroon. After dissection, the proteome from guts of parasite-infected flies were compared to that from uninfected flies in order to identify quantitative and/or qualitative changes associated to infection. A total of 3291 proteins were identified of which 1818 could be quantified. The comparative analysis allowed identifying 175 proteins with significant decreased abundance in infected as compared to uninfected flies, while 61 proteins displayed increased abundance. Among the former are RNA binding proteins, kinases, actin, ribosomal proteins, endocytosis proteins, oxido-reductases, as well as proteins that are unusually found such as tsetse salivary proteins (Tsal) or Yolk proteins. Among the proteins with increased abundance are fructose-1,6-biphosphatase, serine proteases, membrane trafficking proteins, death proteins (or apoptosis proteins), and SERPINs (inhibitor of serine proteases, enzymes considered as trypanosome virulence factors) that displayed highest increased abundance. Sodalis, Wiggleswothia and Wolbachia proteins are strongly under-represented, particularly when compared to data from similar experimentation conducted under controlled conditions on T. brucei gambiense infected (or uninfected) G. palpalis gambiensis insectary reared flies. Comparing the overall recorded data, 364 proteins identified in gut extracts from field flies were shown to have a homologue in insectary flies. Discrepancies between the two studies may arise from differences in the species of studied flies and trypanosomes as well as in differences in environmental conditions in which the two experiments were carried out. Finally, the present study together with former proteomic and transcriptomic studies on the secretome of trypanosomes, on the gut extracts from insectary reared and on field collected tsetse flies, provide a pool of data and information on which to draw in order to perform further investigations on, for example, mammal host immunization or on fly vector competence modification via para-transgenic approaches.

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Microbiota-induced changes in Drosophila melanogaster host gene expression

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