Temperature-dependent dopaminergic disruption by chlorpromazine in the rotifer Brachionus calyciflorus: Evidence from receptor gene expression and population response.

Temperature-induced amplification of drug toxicity brings about mounting ecological risks, but the effects of neuroactive substances like chlorpromazine (CPZ) on non-target aquatic invertebrates under sudden temperature shifts remain unclear. We investigated the temperature-dependent neurotoxicity of CPZ in the freshwater rotifer Brachionus calyciflorus, focusing on dopaminergic signaling. We first identified and functionally validated the dopamine receptor gene BcDopR1 in B. calyciflorus, a receptor featuring a 1371-bp open reading frame (ORF) that encodes a 456-amino acid polypeptide with seven transmembrane domains. Heterologous expression in HEK293T cells showed dopamine significantly elevated intracellular cAMP in BcDopR1-transfected cells, while CPZ dose-dependently inhibited this DA-induced cAMP response, confirming BcDopR1 as a functional D1-like dopamine receptor and CPZ as its potent antagonist. Rotifers were exposed to CPZ (0-250 μg/L) under three ecologically relevant temperatures (18, 25 and 32 °C). The peak BcDopR1 expression was observed in the control group at 25 °C, and CPZ exposure inhibited its expression in a concentration- and temperature-dependent manner. As temperature increased from 18 to 32 °C, rotifers exhibited significant reductions (p < 0.05) in several morphological traits, including lorica length, posterior lateral spine length, body size, and egg size. At the population level, the maximum population density first increased and then decreased, whereas the population growth rate increased significantly (p < 0.05). Within 0-250 μg/L CPZ, rotifers at 18 and 25 °C (except 32 °C) showed a gradual increase in lorica length and body size, whereas their egg size and maximum population density first increased then decreased. These findings highlight the susceptibility of aquatic invertebrates to neuroactive contaminants and the compounding role of thermal stress in amplifying pharmaceutical ecotoxicity. BcDopR1 emerges as a promising molecular biomarker for assessing neuroactive pharmaceutical ecological risks under climate change, and advances understanding of zooplankton adaptation to multiple environmental stressors.