Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

MRNA-Seq of Daphnia magna exposed to abamectin, chlorpyrifos, nicotine, pyriproxyfen and thiacloprid against untreated control groups

ABSTRACT: The experiment investigates how abamectin, chlorpyrifos, nicotine, pyriproxyfen and thiacloprid affect the gene expression in the freshwater invertebrate Daphnia magna. The experiment aimed to distinguish neurotoxic mechanisms and derive mechanistic information relevant for environmental risk assessment. Juvenile D. magna were first subjected to acute immobilization tests following OECD Guideline 202 to establish 48‑hour sublethal effect concentrations for each substance, which were then used as exposure levels in subsequent assays so that molecular changes could be characterized. Pooled animals from the low and high exposure groups and corresponding controls were sampled after 48 hours for whole-animal RNA extraction, and poly(A)-enriched RNA was sequenced on an Illumina platform, followed by alignment to the Daphnia magna reference genome and differential expression analysis using a DESeq2-based workflow with independent hypothesis weighting, data-driven log2 fold-change thresholds, and multiple-testing correction to define robust sets of differentially expressed genes per substance and concentration.

INSTRUMENT(S): see manuscript, NucleoSpin RNA/Protein kit lysis buffer (350 µL per tube; Macherey‑Nagel)., Dell PowerEdge R750; CPU: 2 × Intel Xeon Gold 6346 (32 Cores / 64 Threads); RAM: 188 GB, Illumina NovaSeq 6000, FastPrep‑24 homogenizer (MP Biomedicals)

ORGANISM(S): Daphnia magna

SUBMITTER: Alexandra Loll

PROVIDER: E-MTAB-17707 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The experiment investigates how the azole fungicide prochloraz and the organochlorine insecticide endosulfan affect behavior and gene expression in the freshwater invertebrate Daphnia magna in order to distinguish endocrine from neurotoxic mechanisms and derive mechanistic information relevant for environmental risk assessment. Juvenile D. magna were first subjected to acute immobilization tests following OECD Guideline 202 to establish 48‑hour EC5 and EC20 effect concentrations for each pesticide, which were then used as sublethal exposure levels in subsequent assays so that molecular and behavioral changes could be characterized below overt toxicity. At these sublethal concentrations, swimming activity was quantified in a 24‑well plate setup using automated video tracking to assess concentration-dependent changes in activity time as an organism-level functional endpoint sensitive to both metabolic and neurotoxic disturbance. In parallel, pooled animals from the EC5 and EC20 exposure groups and corresponding controls were sampled after 48 hours for whole-animal RNA extraction, and poly(A)-enriched RNA was sequenced (paired-end, 150 bp) on an Illumina NovaSeq platform, followed by alignment to the Daphnia magna reference genome and differential expression analysis using a DESeq2-based workflow with independent hypothesis weighting, data-driven log2 fold-change thresholds, and multiple-testing correction to define robust sets of differentially expressed genes per substance and concentration. Overrepresentation analysis of Gene Ontology terms using a common detected-gene background then mapped these gene-level changes to biological processes, revealing prochloraz-associated enrichment of lipid, steroid and sterol metabolic pathways and endosulfan-associated enrichment of ion transport and signaling-related processes, thereby providing mode-of-action–consistent toxicogenomic signatures that mechanistically complement the observed behavioral phenotypes.

Project description:Testing for toxicity in a number of aquatic organisms is necessary for risk assessments of substances. Yet, aquatic larvae of the so-called EPT (Ephemeroptera, Plecoptera, Trichoptera) taxa are regularly exposed to several environmental contaminants and have been demonstrated to be extremely vulnerable to a variety of environmental pollutants. These results show that existing toxicity testing can result in an underestimating of the risk for EPT taxa and that more toxicity data using EPT taxonomic representatives are needed. Unfortunately, there is a dearth of standardized test techniques and scant published data, particularly for European EPT species. Our study's objective was to create a testing framework for a variety of endpoints in the mayfly Cloeon dipterum. Due to its high prevalence in local waterbodies and its brief lifecycle of a few weeks in the right environmental conditions, C. dipterum was selected as the test organism. To this end, two chronic toxicity tests with semi-static test design and two media renewal per week were performed. Small larvae in the stage L3 based on the wing pad development described by Cianciara (1976) were used in the tests. Four replicates per test concentration and control containing five individuals per replicate were installed. The emergence was determined daily on working days. The tests were conducted at a temperature of 20 °C (± 1 °C) and the illumination was < 1 µE m2s 1 with a light and dark rhythm of 16:8 h. The physico-chemical parameters pH, oxygen concentration, and oxygen saturation were measured using the multiparameter device WTW Multi 1970i at test start, throughout each media renewal, and at test end. Continuous temperature measurements were taken, and weekly lighting assessments were made. Fipronil was administered to C. dipterum larvae in the long-term exposure experiment at nominal concentrations ranging from 0.038 to 0.60 g/L. This test was conducted for 38 days until all larvae had emerged. In the short-term exposure experiment, C. dipterum larvae were exposed to Fipronil in nominal concentrations between 0.038 and 0.30 µg/L for a test duration of seven days. At test end, the larvae were sampled for transcriptome analysis.

Project description:The aim of this mRNA expression profiling experiment was to screen for ecotoxicogenomic fingerprints in juvenile daphnids (daphnia magna) as aquatic vertebrate non-target model exposed to sub lethal concentrations of Imidacloprid. Imidacloprid is is a popular insecticide widely applied in agriculture and by veterinarians against ectoparasites.The Insecticide Resistance Action Committee (IRAC) classified Imidacloprid after its mode of Action (MoA) in the target organism as a nicotinic acetylcholine receptor (nAChR) competitive modulator (Group 4). The goal is to identify toxicogenomic profiles with predictive character and identify potential molecular key events (KE) explaining upstream adverse effects. This will provide useful information to refine and improve existing adverse outcome pathways (AOP). Furthermore, integrating the obtained profiles for this and other tested chemicals in a collective database will enable us in the future to derive predictions about the ecotoxicological hazard for chemcials with unknown apical effects, based on similarly altered transcriptomic and proteomic profiles. In a modified version of the acute immobilisation test (OECD 202), 50 juvenile Daphnids were exposed to two sub lethal concentrations of Imidacloprid for 48 hours under static conditions. Each test comprised of a low exposure (LE), high exposure (HE) and a negative control (NC) group and was performed in triplicates. At 48 hours after introducing the daphnids into the test solutions, RNA and protein was extracted from living daphnids with NucleoSpin RNA/Protein kit (Macherey-Nagel). RNA quality was assessed with a 2100 Bioanalyzer system (Agilent) before messenger RNA was purified (PolyA selection with TruSeq RNA Library Prep Kit v2) and sequenced on an Illumina HiSeq 4000 System (Illumina) in 50 bp single read mode, producing roughly 30 million reads per sample. Adapter sequences were removed with trimmomatic and sequences were aligned to the D. magna reference genome (daphmag2.4) using STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for count normalization and differential gene expression analysis with DESeq2.

Dataset Information

MRNA-Seq of Daphnia magna exposed to abamectin, chlorpyrifos, nicotine, pyriproxyfen and thiacloprid against untreated control groups

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