mRNA-Seq of Daphnia magna exposed to different concentrations of prochloraz and endosulfan against untreated control groups

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.