Dinotefuran (DIN) is a widely used neonicotinoid insecticide that has been detected in an increasing number of aquatic environments. However, the sublethal effects of DIN on native fish during their early developmental stages remain to be fully elucidated. The present study evaluated the developmental, behavioral, biochemical and transcriptomic responses of Opsariichthys bidens embryos and larvae exposed to three environmental concentrations of DIN (10, 100 and 1000 ng/L). The results evidenced that exposure to 1000 ng/L DIN resulted in a significant reduction in hatching rate at 48 hpf and a significant increase in larval malformation rate at 96 hpf. Behavioral assays revealed that 120 hpf larvae exposed to 100 and 1000 ng/L DIN exhibited a significant reduction in swimming activity. Transcriptomic analysis revealed that circadian rhythm was one of the most consistently enriched pathways in larvae following DIN exposure, with key clock-related genes including per1a, per3 and nr1d1 affected. Furthermore, DIN has been demonstrated to disrupt neuroendocrine and immune-related biomarkers, as evidenced by a decline in dopamine, GABA and IL-1β in larvae exposed to 1000 ng/L DIN, and a reduction in T3 and T4 in larvae from three DIN exposure groups. WGCNA (weighted gene co-exposure network analysis) proceeded to identify gene modules associated with thyroid hormones, dopamine, hatching rate and locomotor traits. This further highlighted links among circadian regulation, endocrine disruption, calcium signalling and behavioral impairment. Collectively, these findings suggest that early-life DIN exposure disrupts a circadian rhythm-centered neuroendocrine network in O. bidens, providing mechanistic evidence for the ecological risk of neonicotinoids to native freshwater fish.
