Ushio, M.; Ozawa, S.; Oka, S.-i.; Sado, T.; Kisero, R. O.; Porter, L.; Matrai, E.; Miya, M.

Biodiversity monitoring is crucial for understanding ecosystem dynamics and species distributions, particularly in the context of anthropogenic impacts and climate change. Cetaceans, as key indicator species of marine ecosystems, face increasing threats from human activities, highlighting the need for effective, non-invasive monitoring methods. In the present study, we developed novel Cetacea-specific primer sets to enhance the detection efficiency of cetacean species through environmental DNA (eDNA) metabarcoding, while minimizing the amplification of non-target vertebrates, such as fish and humans. We retrieved 71 cetacean mitochondrial genomes from a public database and designed 20 candidate primer sets, which were assessed in silico for their specificity and capacity to differentiate cetacean sequences. Four primer sets with the best in silico performance were selected for empirical validation using DNA from tissue samples and eDNA from seawater collected from aquarium pools and Hong Kong coastal waters. All four primer sets effectively amplified cetacean DNA from tissue samples. However, in the aquarium pool tests, three primer sets failed to accurately identify one or more cetacean species due to a lack of interspecific variation within the amplified region. From these, we selected one primer set targeting 268 bp insert length in mitochondrial 12S rRNA region, named Ceta, and applied it to water samples collected from Hong Kong coastal waters, where the iconic Indo-Pacific humpback dolphin (Sousa chinensis) was observed. Ceta successfully detected S. chinensis eDNA while avoiding amplification of non-target species such as fish or humans. Our results demonstrate that Ceta is a reliable tool for cetacean eDNA detection, contributing to cetacean conservation and enhancing our understanding of marine biodiversity.