Role of Bmal1 in mediating the cholinergic system to regulate the behavioral rhythm of nocturnal marine molluscs

The circadian rhythm is one of the most general and important rhythms in biological organisms. In this study, continuous 24-h video recordings showed that the cumulative movement distance and duration of the abalone, Haliotis discus hannai, reached their maximum values between 20:00-00:00, but both were significantly lower between 08:00-12:00 than at any other time of day or night (P P > 0.05). Following the injection of three different concentrations of neostigmine methylsulfate, as an AchE inhibitor, the concentration of Ach in the hemolymph, and the expression levels of nAchR in the cerebral ganglia increased significantly (P P < 0.05). The expression levels of the core diurnal clock Bmal1 over a 24-h period also tended to be high during the day and low at night. First, a co-immunoprecipitation assay demonstrated the binding between Bmal1 and AchE or nAchR. A dual-luciferase gene test and electrophoretic mobility shift assay showed that Bmal1 bound to the promoter regions of AchE and nAchR. Twenty-four hours after silencing the Bmal1 gene, the expression levels of AchE and nAchR decreased significantly compared to those of the dsEGFP and PBS control groups, further showing that Bmal1 mediates the cholinergic system to regulate the behavioral rhythm of abalone. These findings shed light on the endocrine mechanism regulating the rhythmic behavior of abalone, and provide a reference for understanding the life history adaptation strategies of nocturnal organisms and the proliferation and protection of bottom dwelling economically important organisms.