Development and Application of a Species–Specific <scp>eDNA</scp> –Based <scp>qPCR</scp> Assay for Early Detection of the Invasive Ascidian <i>Ascidiella aspersa</i>

The solitary ascidian <i>Ascidiella aspersa</i> is an emerging invasive species in coastal ecosystems worldwide, including Korean waters. We developed and validated a species-specific quantitative PCR (qPCR) assay targeting the mitochondrial COI gene, showing high specificity against 128 non-target taxa and robust performance (Efficiency = 110.1%, <i>R</i> ² = 0.9962, LOD = 12 copies/reaction, LOQ = 598 copies/reaction). The assay was applied to over 300 environmental DNA (eDNA) samples collected across 18 harbors between 2019 and 2021, and to an additional survey in 2022 that targeted four representative harbors (Bieung, Tongyeong, Yangpo, and Jeju) selected from the East, West, South, and Jeju Seas. Seasonal detection patterns showed peak DNA concentrations during summer, coinciding with the reproductive season of <i>A. aspersa</i>. Geographic analysis revealed a broader distribution than previously recorded, including new detections in Jeju Island and eastern ports. These findings highlight the assay's utility for early detection, risk assessment, and surveillance of <i>A. aspersa</i>, supporting its integration into national biosecurity frameworks. This study demonstrates the power of eDNA-based diagnostics for managing marine invasive species through scalable and non-invasive monitoring strategies.