The inaudible impact: physiological stress responses to ultrasonic exposure in the solitary ascidian Ciona intestinalis

Ultrasonic technologies are increasingly deployed in coastal and marine environments for antifouling, sonar, underwater communication, and ballast water treatment; however, their biological effects on sessile invertebrates remain poorly understood. This study investigated the physiological responses of the solitary ascidian Ciona intestinalis to continuous (CUES) and pulsed (PUES) ultrasound exposure at 30 kHz using a biochemical, behavioral, histological, and ultrastructural multi-biomarker approach. Animals were exposed for 15 min, 30 min, 1 h, 2 h, 5 h, and 24 h. Oxidative stress biomarkers assessed in visceral tissue included superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST), acetylcholinesterase (AChE), and the GSH/GSSG ratio. Filter-feeding performance, apoptosis in branchial tissue, and ultrastructural integrity of the coronal organ — a mechanosensory structure on the oral tentacles homologous to vertebrate inner ear hair cells — were also evaluated. CUES induced a time-dependent functional impairment of antioxidant defenses, with progressive suppression of SOD, CAT, and non-Se GPx activities and severe depletion of the reduced glutathione pool. These changes were accompanied by branchial cell death, documented by histochemical markers of early and late apoptosis, and by a significant reduction in filter-feeding performance, consistent with loss of ciliated branchial epithelium. CUES also triggered progressive cholinergic dysregulation, manifested as persistent siphon opening and complete loss of the touch-evoked siphon withdrawal reflex after 24 h. Ultrastructural analysis revealed time-dependent reduction in ciliary coverage of coronal organ sensory hair cells, most severe under CUES. Despite delivering instantaneous peak intensities nearly 38-fold higher than CUES, PUES caused comparatively milder and more transient effects, likely because the intermittent emission pattern limited the accumulation of mechanically induced cellular stress. These findings suggest that the temporal structure of ultrasonic emissions may play a more important role than instantaneous peak intensity in determining biological impact. They also provide an experimental basis for incorporating emission mode into the environmental assessment and eco-design of ultrasonic technologies, supporting the development of devices that minimize unintended effects on non-target benthic organisms.