Effects of neem extract on Artemia franciscana: Insights from high-throughput transcriptomics and phenotypic analysis

Neem-derived biopesticides are increasingly applied in agriculture and have been tested in aquaculture research, yet their effects on non-target aquatic invertebrates remain insufficiently characterized. We evaluated the effect of neem extract on the brine shrimp Artemia franciscana using an integrated ecotoxicological approach combining phenotypic, transcriptomic, and histological analyses. Juvenile A. franciscana exhibited dose-dependent mortality and sublethal abnormalities, with a 24 h median lethal concentration of 292.48 mg/L (95% confidence interval, 257.75-331.89) for mortality and a median effective concentration of 146.36 mg/L (95% confidence interval, 113.04-189.50) for the combined endpoint "abnormal + dead". In adults, males showed greater mortality than females after extended exposure. High-throughput RNA sequencing revealed broad treatment-associated differences in transcript abundance, with juveniles displaying downregulation of detoxification enzymes and chitin biosynthesis genes, alongside enrichment of immune- and cuticle-related gene ontologies. Adults showed transcriptional signatures of stress, including upregulation of heat shock proteins and cytoskeletal components, and suppression of genes involved in energy metabolism. Chitin precursor enzymes were selectively downregulated in males, and altered carbohydrate metabolism was observed in females. Histological analyses showed treatment-associated alterations in the embryo-bearing brood sac region and reduced ovarian area in treated females. Overall, neem exposure was associated with phenotypic, histological, and transcriptomic changes in A. franciscana. These results support the use of combined transcriptomic and histopathological endpoints to characterize responses to plant-derived biopesticides in aquatic arthropods.