Multi-locus environmental DNA markers and taxon-specific primers enhanced detection rate of invasive tilapia

Tilapia are native to Africa. Due to introductions for aquaculture and the ornamental fish trade, several species of tilapia have now spread across five continents, making them some of the most ecologically harmful invasive species. During the early stages of their escape, tilapia are often difficult to detect using traditional fish survey methods. Using environmental DNA (eDNA) methods to monitor tilapia invasion is a promising supplementary approach, but currently no eDNA markers or suitable primers have been developed specifically for tilapia. In this study, we designed four pairs of tilapia-specific primers for use in multiplex PCR coupled with high-throughput sequencing, enabling the simultaneous detection of multiple tilapia species in a single reaction. We found that the taxon-specific primers (Tila-1–4) exhibited higher detection sensitivity compared to the universal primer (tele02), reaching 5.33 × 10 -7 ng/µL. Furthermore, multi-locus amplification reached a 95% limit of detection (LOD) at 2.66 × 10 −6 ng/µL, which was significantly better than 2.3 × 10 -3 ng/µL for single-locus amplification. Finally, we collected water samples from nine rivers across four drainages in China to test the method that we developed. The results indicated that tilapia have invaded river systems such as the Yangtze River and the Pearl River. Specifically, Oreochromis niloticus and Coptodon zillii were the dominant invasive species detected, with some locations also showing the presence of O. aureus and Sarotherodon galilaeus . This method provides an effective tool for early monitoring of tilapia invasion, which may inspire the development of similar strategies for monitoring other invasive species.