Global Gene Expression Profiling in Litopenaeus stylirostris
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ABSTRACT: The primary goal of this project is to monitor host global gene expression patterns in response to viral infection in the shrimp, Litopenaeus stylirostris. Specific Pathogen Free (SPF) L. stylirostris were obtained from High Health Aquaculture (Honolulu, Hawaii) and kept in environmentally controlled tanks. For control, animals were injected with saline (30 ul) between the second and third tergal plates of the lateral side of the tail using a 1 ml tuberculin syringe. Infected individuals were inoculated with homogenate created from IHHNV infected shrimp tissue. After 24 hours, the shrimp were sacrificed and tissue was collected from the ventral and flash frozen in liquid nitrogen and stored in the -80 ºC freezer. Libraries of sequence tags were generated via the Long-SAGE kit (Invitrogen®, Carlsbad, CA) until the ditag PCR preparation step and directly pyrosequenced by 454 Roche.
Project description:Oral susceptibility of Aedes aegypti mosquitoes to dengue viruses varies between different Aedes species and strains. However, the midgut-specific transcriptional profile that may produce this variation is presently obscure and was the subject of our investigation. The variation in active expression between dengue-2 susceptible (SUS) and refractory (REF) mosquitoes was investigated during the first critical 96 hours after infection Transcriptional profiles were mined from respective guts using the serial analysis of gene expression technique (SAGE) and libraries constructed from midguts obtained from mosquitoes that received a dengue-2 infected blood meal (DENV-2), a non infected blood meal (naive) or a 5% sucrose meal (SM). Here we report that variation between DENV-2 infected libraries versus respective naïve libraries revealed very few transcripts that were common and statistically significant in DENV-2 infected libraries. In addition, the expression profiles among libraries displayed up regulation of antisense transcripts especially in the SUS strain. A strong proclivity towards strain-specificity in differential expression was observed, which suggested an exclusive transcription that is likely up-regulated after DENV-2 infection Thirty Aedes aegypti female mosquitoes aged 4-5 days were transferred to 500 ml paper cups and offered a 5% sucrose meal (SM), a naïve blood meal or a dengue-2 (JAM 1409 strain) infectious blood meal, using standard artificial membrane feeders. Fully engorged females were isolated and maintained on a 5% sucrose solution ad libitum at 26oC and relative humidity till dissection
Project description:The host range of African trypanosomes is influenced by innate protective molecules in the blood of primates. A subfraction of human high-density lipoprotein (HDL) containing apolipoprotein A-I, apolipoprotein L-I, and haptoglobin-related protein is toxic to Trypanosoma brucei brucei but not the human sleeping sickness parasite Trypanosoma brucei rhodesiense. It is thought that T. b. rhodesiense evolved from a T. b. brucei-like ancestor and expresses a defense protein that ablates the antitrypanosomal activity of human HDL. To directly investigate this possibility, we developed an in vitro selection to generate human HDL-resistant T. b. brucei. Here we show that conversion of T. b. brucei from human HDL sensitive to resistant correlates with changes in the expression of the variant surface glycoprotein (VSG) and abolished uptake of the cytotoxic human HDLs. Complete transcriptome analysis of the HDL-susceptible and -resistant trypanosomes confirmed that VSG switching had occurred but failed to reveal the expression of other genes specifically associated with human HDL resistance, including the serum resistance-associated gene (SRA) of T. b. rhodesiense. In addition, we found that while the original active expression site was still utilized, expression of three expression site-associated genes (ESAG) was altered in the HDL-resistant trypanosomes. These findings demonstrate that resistance to human HDLs can be acquired by T. b. brucei. Keywords: Trypanosoma, VSG, antigenic switching, HDL-resistance Bloodstream stages of the Lister strain 427 T. b. brucei (MiTat 1.2), expressing VSG221, were used in these studies. Cells were cultured in HMI-9 medium with the addition of heat inactivated fetal bovine serum (FBS) (10%) and Serum Plus (10%). T. b. brucei 427-221 is an antigenically stable line and contains a single copy of the vsg221 gene within the 221 expression site (221ES). At a cell density of approximately 1,000,000 cells/ml, T. b. brucei 427-221 were exposed to various amounts of human HDLs for 24 h in a 6 well plate. Surviving trypanosomes were counted using a hemocytometer then diluted into fresh HMI-9 medium and allowed to recover for 5-14 days. Once the cells had grown to a density of approximately 1,000,000 cells/ml, they were once again incubated with human HDLs. Each round of selection was performed with increasing concentrations of human HDLs and freezer stocks were prepared for each surviving population. Over nine months we conducted eight rounds of human HDL selection, resulting in a population of T. b. brucei that survived incubation with 800 μl of human HDLs (160 lytic U).
Project description:The abundant and widespread coccolithophore Emiliania huxleyi plays an important role in mediating CO2 exchange between the ocean and the atmosphere through its impact on marine photosynthesis and calcification. Here, we use long serial analysis of gene expression (SAGE) to identify E. huxleyi genes responsive to nitrogen (N) or phosphorus (P) starvation. Long SAGE is an elegant approach for examining quantitative and comprehensive gene expression patterns without a priori knowledge of gene sequences via the detection of 21-bp nucleotide sequence tags. E. huxleyi appears to have a robust transcriptional-level response to macronutrient deficiency, with 42 tags uniquely present or up-regulated twofold or greater in the N-starved library and 128 tags uniquely present or up-regulated twofold or greater in the P-starved library. The expression patterns of several tags were validated with reverse transcriptase PCR. Roughly 48% of these differentially expressed tags could be mapped to publicly available genomic or expressed sequence tag (EST) sequence data. For example, in the P-starved library a number of the tags mapped to genes with a role in P scavenging, including a putative phosphate-repressible permease and a putative polyphosphate synthetase. In short, the long SAGE analyses have (i) identified many new differentially regulated gene sequences, (ii) assigned regulation data to EST sequences with no database homology and unknown function, and (iii) highlighted previously uncharacterized aspects of E. huxleyi N and P physiology. To this end, our long SAGE libraries provide a new public resource for gene discovery and transcriptional analysis in this biogeochemically important marine organism. Keywords: Emiliania, gene expression, nutrients, SAGE, phosphate, nitrogen Emiliania huxleyi CCMP 1516 was obtained from the Provasoli-Guillard Center for the Culture of Marine Phytoplankton, Bigelow Laboratories. Cultures were grown at 18°C on a 14 h:10 h light:dark cycle (140 µmol quanta m-2 s-1). Nitrogen and phosphate replete (Replete: 35 µM NO3- and 1.5 µM PO43-), low nitrogen (-N: 10 µM NO3-) and low phosphate (-P: 0 µM PO43-) cells were grown in f/50 medium without Si. Locally collected seawater was filtered (pore size, 0.2 µm) and autoclaved. Filter-sterilized inorganic nutrients, trace metals and vitamins (thiamin, biotin and B12) were added after autoclaving. The cells were grown in 8 L batch cultures. The growth of cultures was monitored daily by cell number counted on a hemacytometer, and by relative fluorescence using a Turner Designs AU Fluorometer. Replete cells were harvested in mid-log phase while –N and –P cells were harvested at the onset of stationary phase for SAGE analysis.
Project description:Nitric oxide (NO) is a gaseous intercellular signaling molecule that also plays a role in host-parasite relations. NO acts rapidly, either via regulation of soluble guanylate cyclase, or by direct interactions with enzymes and other proteins, and has also been shown to have effects on gene expression. Here, we use SAGE (Serial Analysis of Gene Expression) to identify NO-responsive changes in gene expression in Schistosoma mansoni following a 3 hour exposure to sodium nitroprusside, an NO donor. Overall, these results indicate that NO does not rapidly induce large-scale changes in schistosome gene expression, but that expression of particular genes of interest appear to respond to NO. Keywords: Schistosoma, SAGE, NOS, nitric oxide, gene expression Adult S. mansoni perfused from infected Swiss-Webster female mice (obtained from the NIAID Schistosomiasis Resource Center) 42-49 days postinfection were maintained in culture (RPMI medium) overnight and then exposed for 3 hours to either 1 mM sodium nitroprusside (SNP), a well-characterized NO donor, or to RPMI alone. Worms remained viable and motile following treatment. Total RNA was extracted with Trizol (Invitrogen) and treated with DNAse 1 (Ambion) to remove contaminating genomic DNA, and Long-SAGE libraries constructed.
Project description:Biomphalaria glabrata infection by the Schistosoma mansoni free-swimming miracidium and its subsequent development to the parasitic sporocyst stage is critical to establishment of viable infections and triggers a variety of physiological, biochemical and molecular changes. Here, we describe a genome-wide analysis of the S. mansoni miracidium and developing sporocyst. Keywords: life-cycle, development, host-interaction We generated transcriptomic profiles of the developing larval stages of Schistosoma mansoni using long serial analysis of gene expression (LongSAGE). Five cDNA libraries were constructed from miracidia and in vitro cultured 6- and 20-day old sporocysts maintained in sporocyst medium (SM) or in SM conditioned by previous cultivation with cells of the B. glabrata embryonic (Bge) cell line. From five libraries, 314,799 SAGE tags were sequenced and resulted in a total of 21,440 unique sequence tags. A total of 254 tags were differentially expressed during “conditioned” development and 236 tags were differentially expressed during “un-conditioned” development. In addition, 53 tags were found to be differentially expressed between 6-day conditioned and unconditioned sporocysts and 42 tags between 20-day conditioned and unconditioned sporocysts.
Project description:The transcriptomic response of two strains of the Pacific whiteleg shrimp, different in their resistance to Taura Syndrome Virus (TSV), in response to infection with TSV and Yellow Head Virus (YHV). Changes in gene expression in the shrimp’s hepatopancreas were assessed using a cDNA microarray containing 2,469 putative unigenes. The patterns of gene expression between the shrimp strains were considerably similar, except for the more advanced stages of Taura Syndrome. Between the different treatments approximately 250 genes were differently expressed. The most advanced stages of YHV infection showed the highest number of differently expressed genes. During infection there were profound changes in the expression of genes related to lipid and protein metabolism, cellular trafficking, immune defense and stress response. Keywords: Disease state analysis, disease resistance There were 5 biological replicates for each of the groups in this experiment. Also, two strains of Litopenaeus vannamei were used: a strain resistant to TSV and a strain susceptible to TSV (Kona line). The treatments consisted of injecting both strains with 60mL of a shrimp extract made from shrimp previously injected with either a SPF shrimp extract (1x10-4), Taura Syndrome Virus (1x10-5) or Yellow Head Virus (1x10-4). The 2 initial control groups were composed of hepatopancreas samples from both strains prior the injections. Samples were also collected from at days 1 and 2 from both strains from the 3 different treatments (control, TSV and YHV).
Project description:Adult male grass shrimp were exposed for 96 hours to LC50 concentrations of either Fipronil, Endosulfan, or Cadmium, as well as a Carrier Control exposure. RNA was extracted from whole-body homogenates using the RNABee kit. Tags were clustered to identify tags diagnostic of the different exposures. Keywords: SAGE, Grass shrimp, ecotoxicogenomics 3 randomly selected shrimp were pooled for each library. Libraries were constructed using the I-SAGE long kit from Invitrogen.
Project description:Comparisons of gene expression profiles between ovaries of before (day 0) and after eyestalk ablation (days 1, 4 and 7) of domesticated 14-month-old black tiger shrimp (Penaeus monodon) were made using a cDNA microarray. Differentially expressed genes were identified through the microarray analysis, and the microarray results were confirmed by real-time PCR. Selected genes were further characterized. A cDNA microarray consisting of 5,568 features was constructed from EST libraries of P. monodon. RNA samples were extracted from the ovaries of before (day 0) and after eyestalk ablation at days 1, 4 and 7 from shrimp from the Shrimp Genetic Improvement Center, Thailand. The RNA was converted into cDNA by indirect aminoallyl-cDNA labelling method (LabelStar Array, Qiagen). aa-cDNA from day 0 samples were coupled with Cy3 dye as a reference, and those from days 1, 4 and 7 were coupled with Cy5 dye.
Project description:Shellfish processing workers are highly susceptible to respiratory illnesses such as allergies and asthma. However, the airborne biological exposures in this industry are not well characterized. This study aimed at identifying and quantifying airborne biological exposures in the shrimp processing industry and assessing their impact on human health. Our findings show that shrimp processing workers are exposed to several allergenic proteins and irritants. High levels of the major shrimp allergen tropomyosin were detected, with the cooking and peeling departments identified as high-exposure areas. Moreover, workers had a high prevalence of respiratory symptoms and elevated levels of selected biomarkers of asthma and allergy which correlated with the measured total airborne protein levels in their work environment. Our study provides important novel evidence showing the occupational burden of airborne biological exposures in the shrimp industry and identifying critical work task. Altogether, the results underscore the need for improved targeted protective measures.
Project description:Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive fibrosing interstitial lung disease that is unresponsive to current therapy. While it carries a median survival of less than 3 years its rate of progression varies widely between patients. We hypothesized that studying the gene expression profiles of physiologically stable patients and those in which the disease progressed rapidly after the initial diagnosis would aid in the search for biomarkers and contribute to the understanding of disease pathogenesis. We generated 12 Idiopathic Pulmonary Fibrosis (IPF) lung parenchyma SAGE profiles. Initial cluster analysis including 8 other public available lung SAGE libraries verified that the IPF transcriptome is distinct from normal lung tissue and other lung diseases like COPD. In order to identify candidate markers of disease progression we segregated the IPF SAGE profiles in two groups based on clinical parameters regarding lung volume and lung function.