Monitoring of multiple fish species by quantitative environmental DNA metabarcoding surveys over two summer seasons

Periodic monitoring can provide important information for the protection of endangered fish, sustainable use of fishery resources and management of alien species. Previous studies have attempted to monitor fish using non-invasive environmental DNA (eDNA) technology, generally employing quantitative PCR to quantify the eDNA concentration. However, the throughput was limited. High-throughput metabarcoding technology can detect the DNA of multiple species simultaneously in a single experiment but does not provide sufficient quantification. In this study, we applied a quantitative metabarcoding approach to simultaneously quantify the eDNA concentration of an entire fish assemblage in a small reservoir over two summer seasons. Traditional surveys were also conducted to investigate the individuals of fish. The eDNA concentrations were quantified using quantitative metabarcoding, and the fish species detected using this approach were highly consistent with the results of traditional fish monitoring. A significant positive relationship was observed between the eDNA concentration and fish species abundance. Seasonal changes in fish community structure were estimated using eDNA concentrations, which may reveal the activity seasons of different fish. The eDNA concentrations of different fish species peaked at different water temperatures, reflecting the differential responses of fish species to this environmental factor. Finally, by detecting outlier eDNA concentrations, the spawning activities of 13 fish species were estimated, 12 of which were roughly consistent with the current knowledge of fish spawning periods. These results indicate that quantitative eDNA metabarcoding with dozens of sampling times is useful for the simultaneous ecological monitoring of multiple fish species.