Project description: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.
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:The transcriptional response of H. armigera larvae was analyzed after feeding on gossypol supplemented diet, a toxic secondary metabolite produced in cotton plants, to detect potential detoxification enzymes possibly involved in detoxification of gossypol by H. armigera. A one-color microarray-based gene expression analysis was performed with Cy3 labeled cRNA of gut and rest of the body of H. armigera larvae that fed on 0.016%, 0.16% gossypol and control diet. For each treatment and tissue four biological replicates were used.
Project description:The transcriptional response of H. virescens larvae was analyzed after feeding on gossypol supplemented diet, a toxic secondary metabolite produced in cotton plants, to detect potential detoxification enzymes possibly involved in detoxification of gossypol by H. virescens. A one-color microarray-based gene expression analysis was performed with Cy3 labeled cRNA of gut and rest of the body of H. virescens larvae that fed on 0.32% gossypol and control diet. For each treatment and tissue four biological replicates were used.
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:We found that the midgut shows striking regional differentiation along its anterior-posterior axis. Ten distinct subregions differ in cell morphology, gene expression and aspects of Notch signaling. RNA from isolated regions that was analyzed by RNAseq revealed spatially regulated expression of hundreds of enzymes and other genes with likely tissue functions. 10 midgut segments comprising from 1-3 subregions x 3 replicates from each segment = 30 samples
Project description:Among the diverse forms of symbioses, facultative nutritional mutualism forged by the host and its resident gut microbiota permits the symbiont to adapt to the changing nutritional environment during the host’s life time. The horizontally acquired gut bacteria in Drosophila are a perfect example of nutritional mutualists. Here, we study the Lactobacillus plantarum (Lp WJL) infection effect in the Drosophila Genetic Reference Panel (DGRP) collection in context of larvae raised in chronic undernutrtion.
Project description:This study investigated how gut-derived bacterial supplementation influences the honey bee gut microbiota, metabolism, and proteome under three experimental settings: (i) controlled laboratory conditions (C versus B), (ii) semi-controlled laboratory conditions with or without social interaction with older nestmates (COB versus BOB), and (iii) field conditions (CON versus TRT). A bacterial mixture containing Lactobacillus helsingborgensis, L. apis, Bifidobacterium choladohabitans, and B. polysaccharolyticum was administered. Gut samples were collected after 10 days and analyzed for microbiome (16s rRNA sequencing), metabolome (1H NMR), and proteome profiles (LFQ-proteomics, laboratory experiment only). Proteomic profiling revealed distinct group-specific host responses. Control bees (C and COB) upregulated ribosomal proteins linked to protein synthesis and cellular stress. Supplemented bees in group B showed strong induction of major royal jelly proteins (MRJP1 and MRJP5), associated with neural modulation and colony behavior. In BOB, elevated expression of mitochondrial enzymes suggested improved energy metabolism in the presence of social context. Bacterial supplementation induces reproducible, condition-dependent changes across microbiota, metabolic, and proteomic layers. These findings provide mechanistic insight into how microbial interventions reshape gut function and support honey bee health under realistic environmental conditions.
Project description:Transcriptional responses in the gut of the main malaria vector Anopheles gambiae following oral bacterial infection with the entomopathogen Serratia marcescens were identified using DNA microarrays. S. marcescens is a common member of the mosquito gut microbiota, found in both laboratory reared and field collected mosquitoes, that can be potentially pathogenic as in Drosophila (Nehme et al., 2007), while it has been shown to influence the outcome of Plasmodium infections (Bando et al., 2013). S. marcescens belongs to the Enterobacteriaceae family, members of which have been shown to influence malaria transmission dynamics (Cirimotich et al., 2011, Boissiere et al., 2012). To further investigate the interactions between S. marcescens and the mosquito host, likely to shape, directly or indirectly, malaria transmission dynamics, An. gambiae mosquitoes, from the recently established N'gousso M form laboratory colony that retains much of the genetic variation of field mosquitoes, were antibiotic treated for 5 days and subsequently orally infected with the Db11-GFP strain of S. marcescens. Bacteria-fed mosquitoes were selected 2 days post infection, and, 3 days post infection, guts from bacteria-fed mosquitoes were dissected. Uninfected control mosquitoes were treated in the same way. Differential expression in the gut of S. marcescens infected mosquitoes, compared to uninfected controls, was identified by hybridizing labelled complementary RNA, derived from total RNA extracted from the respective gut pools, in customized Agilent 4x44k gene expression microarrays, comprising oligonucleotide probes encompassing all An. gambiae annotated genes of the AgamP3.6 release, with each probe represented in duplicate.