Monitoring Alien Species Diversity in Ballast Water Based on Environmental DNA Metabarcoding

Invasive alien species pose serious threats to ecosystems, public health, and socio-economic systems, with ballast water discharge serving as a major pathway for their introduction. Organisms that survive the harsh conditions inside ballast tanks may establish populations in recipient ports, where they can disrupt native biodiversity. Therefore, effective monitoring of ballast water is essential for reducing the risk of biological invasions. This study applied environmental DNA (eDNA) metabarcoding to investigate the biological communities in ballast water from ships arriving at Dongjiakou Port, Qingdao, with a particular focus on alien species. Three universal primer sets targeting the 18S V4, 18S V9, and 12S regions were used to amplify and sequence DNA from phytoplankton, invertebrates, and fish. In total, nine ballast water samples were collected from six ships originating from the South China Sea, Seto Inland Sea, Taiwan Strait, and the Yellow Sea. Bioinformatic analysis revealed 16 alien phytoplankton species, 13 alien invertebrate species, and 12 alien fish species, including three invasive species: Styela clava, Lates calcarifer, and Anguilla anguilla. Species composition varied considerably among tanks on the same ship, whereas ballast water location and age had no significant effect on composition across different ships. These results demonstrate the potential of eDNA metabarcoding as an efficient, noninvasive approach for monitoring ballast water biodiversity and alien species. Such insights are valuable for informing policy and management strategies to curb the spread of invasive species through shipping networks.