Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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MRNA-Seq of Daphnia magna exposed to different concentrations of sucralose against untreated control groups

ABSTRACT: The experiment investigates how the artificial sweetener sucralose affects behavior and gene expression in the freshwater invertebrate Daphnia magna in order to derive mechanistic information relevant for environmental risk assessment. Juvenile D. magna were first subjected to a behavioral swimming activity test based on the acute immobilization test (OECD Guideline 202). As no swimming activity alteration or acute effect was observed, the main test including transcriptomics, was conducted with 10 µg/L and 100 mg/L as test concentrations. Swimming activity was quantified in a 24‑well plate setup using automated video tracking. In the main test, pooled animals from each exposure group 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.

INSTRUMENT(S): 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-16759 | 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: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.

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MRNA-Seq of Daphnia magna exposed to different concentrations of sucralose against untreated control groups

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