RNA-seq of Daphnia magna exposed to prochloraz in a 3-generation experiment
Ontology highlight
ABSTRACT:
INSTRUMENT(S): BGISEQ-500
ORGANISM(S): Daphnia magna
SUBMITTER: Rikke Poulsen
PROVIDER: E-MTAB-9309 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Custom D. magna gene expression microarray (Design ID: 023710, Agilent Technologies)were used to characterise gene expression profiles of Daphnia magna neoantes exposed to silver nanoparticles ( AgNPs ) or silver nitrate ( AgNO3 ) for 24 hours.
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Project description:Study of the tetrapod limb has contributed a great deal to our understanding of developmental pathways and how changes to these pathways affect morphology. Most data on tetrapod limb development is known from amniotes, with far less known about genetic mechanisms of limb development in amphibians. To better understand the mechanisms of limb development in anuran amphibians, we use cyclopamine to inhibit Hedgehog signaling at various stages of limb development in Xenopus. We use transcriptomic analysis following cyclopamine exposure to understand the downstream effects of Hedgehog inhibition on gene expression. We find many aspects of Hedgehog function appear to be conserved with respect to amniotes, including the responses of ptc genes, gremlin, bmp2, and the autoregulatory property of shh. We show that, as was proposed based on experiments in chick, Sonic hedgehog plays two distinct roles in limb development â specification of digit number and specification of digit identity. In contrast to these points of conservation, we find that Hedgehog signaling is required for the maintenance of early limb bud outgrowth in Xenopus, a requirement not known for any other tetrapod. Experiment Overall Design: Eight microarray experiments were performed using RNA extracted from batches of hindlimbs dissected from Xenopus laevis tadpoles. Four of these were experimental groups exposed to cyclopamine at 1 µg/ml for 24 hours before animal fixation, and four were experimental groups exposed to the ethanol, the cyclopamine vehicle, at the same concentration used for cyclopamine vehicle for 24 hours. Each pool of RNA consisted of material from 18-22 hindlimb buds. Two experimental groups and two control groups were from one clutch of tadpoles, and the other four pools were from another clutch. Both samples within each of four paired groups (experimental vs. control) were exposed and processed simultaneously.
2008-10-18 | E-GEOD-8293 | biostudies-arrayexpress
Project description:Variation in gene expression is known to be important for morphological evolution, however little is known about its general propensity. Here we examine a pair of frogs M-bM-^@M-^S Xenopus laevis and Xenopus tropicalis M-bM-^@M-^S with highly similar embryology, and ask how their transcriptomes compare. Despite separation for over ~30-90 million years we found a strong conservation in gene expression in the vast majority of the expressed orthologs. There were a significant number of changes in the level of expression of genes. Changes in timing of expression, heterochrony, were much less common, and were often found in genes and pathways that reflect responses to selective features of the environment. Differences in gene expression levels were concentrated in the earliest embryonic stages. We propose that different evolutionary rates across developmental stages may be explained by the stabilization of cell fate determination pathways in later stages. 96 microarrays, across developmental stages in Xenopus laevis and Xenopus tropicalis in wildtype embryos. For each of the two timecourse indepenent triplicates (clutches) were generated.
2011-06-01 | E-GEOD-27227 | biostudies-arrayexpress
Project description:Pesticides are continuously released into the environment, with possible long-term consequences on aquatic organisms. One of the pesticides still applied in several crops in some countries is the fungicide carbendazim, ending up in surface waters with concentrations reaching 5 µg/L. Daphnia magna (clone k6) was used in this study as a model organism and it was exposed to a sub-lethal concentration of carbendazim (5 µg/L) for twelve generations. Gene expression alterations induced by this compound were assessed in the F0 and F12 generations using D. magna custom microarrays. Results revealed that carbendazim caused changes at the gene expression level in both generations. Genes involved in response to stress, DNA replication/repair, neurotransmission, protein biosynthesis, ATP production, lipids and carbohydrates metabolism were the most affected in both F0 and F12, although a lower number of differentially expressed genes were observed in the F12 generation exposed to carbendazim. The exposure of daphnids to carbendazim did not cause a stable change in gene expression from F0 to F12 generations. Effects at the gene expression level were early detected at the F0 generation after a short-time exposure (10 days), highlighting the advantages of using high throughput tools as early warning analysis but also providing information on chemical mode of action, which can add value in risk assessment procedures. For the microarray experiment, neonates with less than 24h were picked from the F0 and F12 generations from both clean medium and carbendazim. Three replicates per each treatment were used and consisted of 5 daphnids aging 10 days old. A total of three replicates per treatment were used and each biological replicate was individually hybridized on the array. A single-color design was used, using the Agilent one-color RNA Spike-In Kit (AgilentTechnologies, Santa Clara, CA, USA), following the manufacturers protocol.
2017-05-01 | E-GEOD-78120 | biostudies-arrayexpress
Project description:Assessing the risks of long-term exposure to low doses of pharmaceuticals is an identified research need, particularly for those that may act as neural disruptors in non-vertebrate species. Selective serotonin reuptake inhibitors (SSRIs) act by blocking the re-uptake of serotonin in the nerve synapses, increasing the effective concentration of serotonin in the intra-synaptic space and therefore stimulating serotoninergic neurons. This effect is used worldwide to treat clinical depression in humans, with the consequence of their widespread release into the environment. SSRIs have been found to alter the reproductive physiology of D. magna and other invertebrates in a biphasic way. Low levels of fluoxetine stimulated offspring production in Daphnia magna and Ceriodaphnia dubia at 36 and 50 µg/l, respectively, but higher exposure levels inhibited reproduction in the same species. In this study we explore the hypothesis that SSRI can affect D. magna juvenile developmental rates and offspring production disrupting serotonin activity that is known to regulate carbohydrate and oxidative metabolic pathways. The primary mode of action was tested using transcriptomic analyses of juveniles and adults exposed to SSRIs (fluoxetine 80 ppbs, fluovoxamine 30 ppbs) and serotonin-immunocytochemistry assays of D. magna brains. Reproduction, respirometry and biochemical measurements allowed to related gene and immunological base effects to phenotypic responses.
2013-03-15 | E-GEOD-45053 | biostudies-arrayexpress
Project description:Environmental and Genetic Modifiers of squint Penetrance during Zebrafish Embryogenesis. Gene expression changes due to temperature shift during devlopment in homozygous squint mutants and wild_type embryos
2006-07-24 | E-MEXP-775 | biostudies-arrayexpress
Project description:Microarray Construction The D. magna microarray was constructed using DNA fragments from three sources: i) Stress-specific cDNAs were generated using Selective Subtractive Hybridisation (SSH) on organisms exposed to the stressors shown in Table 1. SSH was performed with forward and reverse subtraction using a PCR-Select cDNA Subtraction Kit (Clontech Laboratories Inc, USA). cDNA was prepared from mRNA extracted from batches of 100 D. magna, 48 h old, using a Straight AM-CM-"M-BM-,s mRNA Isolation Kit System (Novagen Inc., UK) (2,029 clones). ii) ESTs obtained from a cDNA library from unexposed mixed aged organisms obtained from a 3-4 week old culture (Watanabe et al. 2005) (10,272 clones). iii) cDNAs isolated following an SSH, as above, between 15 adults carrying eggs and 75 juveniles (Soetaert et al. 2006) (1,143 clones). Gene fragment annotation Sequencing of the 3,172 SSH ESTs was carried out with M13-Long primers. Blast searches were performed on specific fragments that responded significantly to the exposure treatment. Sequences were annotated according to BlastX homology search against Uniprot Swissprot (http://www.ebi.ac.uk/swissprot) and Uniprot Trembl (invertebrate section, http://www.ebi.ac.uk/trembl) March 2006. Sequences were only annotated if they were found to have a Blast hit with the expect value smaller than 0.00001 and a score above 50. GeneBank/UniProt Accession number and speciesM-CM-"M-BM-, match were recorded with each annotation. We printed PCR products generated from the above, onto glass slides. All spots have been sequenced except for (iii), only a few available and some sequencing failed for various reasons. Therefore we have the sequences for around 90% of the spots. BLAST searches revealed a large proportion (nr 60%) had no significant match with existing entries in GenBank/Uniprot/Trembl. Because of low confidence we've not considered them, otherwise this would lead to huge and erroneous assumptions. All (i and ii) sequences are available either on GenBank or on our website. All the spots that came out as significant on our experiments were reviewed and only those that have been annotated, with significant BLAST match, are being published, with respective Accession numbers, species match and E-values. Fragments were therefore described as M-CM-"M-BM-,SunnanotatedM-CM-"M-BM-,M-CM-= where no significant difference was measured or M-CM-"M-BM-,Sno significant matchM-CM-"M-BM-,M-CM-= for those that were significant but no matches were found. Significant genes were annotated respectively as described above. Dose Reponse Tests were carried out using the developed microarray on cadmium, ph, lufenuron, calcium limitation and ibuprofen; details below: Cadmium: RNA was obtained from populations of 240 D. magna, 24-48 h old, exposed for 24 h to sublethal concentrations of cadmium chloride from 0, 10, 33 to 60 M-CM-^BM-BM-5g L-1 (corresponding to 0, 6.13, 20.24 and 36.79 M-CM-^BM-BM-5g L-1 Cd2+), in quadruplicates. pH: RNA was obtained from populations of 240 D. magna, 24-48 h old, exposed for 24 h to pH ranging from 8.0 (controls), 5.5, 5.3 and 5.2, in quadruplicates. pH was maintained by adding HCl on a daily basis. Lufenuron: RNA was obtained from populations of 240 D. magna, 24-48 h old, exposed for 24 h to sublethal concentrations of C14 labelled, lufenuron ranging from 0, 0.35, 0.59 to 1.00 ug L-1, in quadruplicates. Calcium: RNA was obtained from populations of 240 D. magna, 24-48 h old, exposed for 24 h to calcium chloride limitation ranging from 195.87 (culture condition), 20, 10 and 5 mg L-1 (CaCo3), in quadruplicates. Ibuprofen: RNA was obtained from populations of 300 D. magna, 24-48 h old, exposed for 24 h to ibuprofen (IB) sodium; 0 (control), 20, 40 and 80 mg L-1, in *quadruplicates. *One replicate at 80 mg L-1 was lost, thus this treatment was done in triplicate. IB was measured as the pharmacological active ingredient. Juvenile D. magna were used in order to restrict the response to genes associated with stress responses and growth, rather than genes associated with different stages of reproduction. Following exposure, daphnids were stored in RNAlater (Ambion, UK) at -80M-CM-^BM-BM-0C.
2007-11-05 | E-MAXD-20 | biostudies-arrayexpress
Project description:The aim of this study was to measure chronic sublethal effects of the flame retardant Tris(2-butoxyethyl)phosphate (TBEP) on gene expression, associated protein activity and life-history endpoints (reproduction, development) in the freshwater crustacean Daphnia magna. We exposed <24h old D. magna neonates for 21 days to culture medium (control) and to 3 sublethal doses of TBEP: 14.7, 147 and 1470 M-BM-5g/L. Exposure doses were chosen from 48h acute toxicity testing and represent 1/10000, 1/1000 and 1/100 of the LC50, respectively. Each condition (control and the 3 different doses) were performed on 3 independant biological replicates, with 10 individuals each. We used a custom 15000 sequences D. magna microarray to measure transcriptional effect of each of the TBEP doses in comparison to unexposed controls. We exposed <24h old Daphnia magna to culture medium (unexposed controls) and 3 sublethal doses of TBEP (14.7M-BM-5g/L, 147M-BM-5g/L and 1470M-BM-5g/L) for 21 days. We used 3 biological replicates of 10 individuals for each condition.
2014-02-19 | E-GEOD-55132 | biostudies-arrayexpress
Project description:Prochloraz is a fungicide known to cause endocrine disruption through effects on the hypothalamic-pituitary-gonadal (HPG) axis. To determine the short-term impacts of prochloraz on gene expression and steroid production, adult female fathead minnows (Pimephales promelas) were exposed to the chemical (0 or 300 M-NM-<g/L) for a time-course of 6, 12 and 24 h. Consistent with inhibition of cytochrome P450 17M-NM-1-hydroxylase/17,20-lyase (CYP17) and aromatase (CYP19), known molecular targets of prochloraz, plasma 17M-NM-2-estradiol (E2) was reduced within 6 hours. Ex vivo E2 production was significantly reduced at all time-points, while ex vivo testosterone (T) production remained unchanged. Consistent with the decrease in E2 levels, plasma concentrations of the estrogen-responsive protein vitellogenin were significantly reduced at 24 h. Genes coding for CYP19, CYP17, and steroidogenic acute regulatory protein were up-regulated in a compensatory manner in ovaries of the prochloraz-treated fish. In addition to targeted quantitative real-time polymerase chain reaction analyses, a 15k feature fathead minnow microarray was used to determine gene expression profiles in ovaries. From time-point to time-point, the microarray results showed a relatively rapid change in the differentially expressed gene (DEG) profiles associated with the chemical exposure. Functional analysis of the DEGs indicated changes in expression of genes associated with cofactor and coenzyme binding (GO: 0048037 and 0050662), fatty acid binding (GO:0005504) and organelle organization and biogenesis (GO: 0006996). Overall, the results from this study are consistent with compensation of the fish HPG axis to inhibition of steroidogenesis by prochloraz, and provide further insights into relatively rapid, system-wide, effects of a model chemical stressor on fish. RNA samples for each treatment/time point combination were used for microarray analysis (total of 23 arrays). For the microarray work, total RNA was isolated from ovary samples using RNeasy kits (Qiagen, Valencia, CA, USA). The RNA quality was assessed with an Agilent 2100 Bioanalyzer (Agilent, Wilmington, DE, USA) and quantity was determined using a NanodropM-BM-. ND-1000 spectrophotometer. Total RNA was stored at -80oC until analyzed with oligonucleotide microarrays. Microarray analysis was conducted at the US Army Engineer Research and Development Center (Vicksburg, MS, USA) using a 15k feature fathead minnow microarray (GEO: http://www.ncbi.nlm.nih.gov/geo/; Accession platform number GPL9248) designed by Dr. Nancy Denslow (University of Florida, Gainesville, FL, USA) and manufactured by Agilent Technologies (Palo Alto, CA, USA). The Agilent one-color microarray hybridization protocol (One-Color Microarray-Based Gene Expression Analysis, version 5.7, Agilent Technologies) was used for microarray hybridizations. One M-BM-5g of total RNA was used for all hybridizations. The cDNA synthesis, cRNA labeling, amplification and hybridization were performed following the manufacturerM-bM-^@M-^Ys kits and protocols (Quick Amp Labeling kit; Agilent Technologies). An Axon GenePixM-BM-. 4000B Microarray Scanner (Molecular Devices Inc., Sunnyvale, CA, USA) was used to scan microarray images at 5 M-NM-<m resolution. Data were resolved from microarray images using Agilent Feature Extraction software (Agilent Technologies). Statistical analyses were conducted using Statistica 8 (StatSoft Inc., Tulsa, OK, USA) and GraphPad Instat v. 3.01 (GraphPad Software, San Diego, CA, USA). Data were tested for normality and homogeneity of variance. When data conformed to parametric assumptions, data were analyzed using a parametric one-way ANOVA with chemical treatment as the independent variable. When data did not conform to parametric assumptions, they were either transformed (log 10) or analyzed using a non-parametric KruskallM-bM-^@M-^SWallis test (pM-bM-^IM-$ 0.05). The data are presented as means with standard errors of the mean. Differences were considered significant at p M-bM-^IM-$ 0.05. Microarray data were imported into the Rosetta Resolver 7.2 system for gene expression analysis (Rosetta Biosoftware, Kirkland, WA, USA). Data were normalized using the default, text loader, intensity profile builder settings in the Rosetta Resolver system. Significantly DEGs were identified using one-way ANOVA (p < 0.01) with no multiple testing correction
2011-02-03 | E-GEOD-26958 | biostudies-arrayexpress
Project description:This experiment was conducted to study the short-term (12h) transcriptional responses in Daphnia magna after exposure to the anti-sea lice chemical emamectin benzoate (EMB). The microarray results were further vefiried using qPCR. The gene exression responses were linked to adverse effects after 48h exposure, in order to supply knowledge for environmental hazard assessment of this chemical in non-target crustaceans. Neonatal (<24h) Daphnia magna were exposed to 7.8-2000 pM waterborne emamectin benzoate for 12h. Microarray analysis was performed using pooled whole-organism D. magna (8 individuals) and 4 biological replicates were analyzed for each treatment group.
2016-06-30 | E-GEOD-83859 | biostudies-arrayexpress