Quantifying the influence of salinity and density on environmental DNA shedding and decay rates from turtles

Environmental DNA (eDNA) analysis provides a promising non-invasive approach for monitoring elusive species, such as turtles. However, its effective application requires a comprehensive understanding of eDNA shedding and decay dynamics, which remain poorly characterized for hard-shelled reptiles. In this study, we quantified the eDNA shedding and decay rates of the invasive red-eared slider ( Trachemys scripta elegans ) using controlled microcosm experiments under different slider densities (1, 3, and 6 individuals per tank) and salinity levels (0‰, 5‰, and 15‰). A novel species-specific droplet digital PCR (ddPCR) assay targeting the mitochondrial 12S rRNA gene of the slider was developed and validated. The measured eDNA shedding rates for sliders ranged from 10 4 to 10 6 copies/h. A significant positive correlation was found between slider density and eDNA shedding rate at 0‰ and 5‰ salinity; however, this correlation was absent at 15‰. The positive effect of salinity on shedding was also density-dependent, becoming non-significant at high density. The eDNA decay rate constants varied from 4.30×10 -3 to 1.09×10 -2 /h and exhibited a strong negative correlation with salinity as density increased. Significant interactions between density and salinity were observed for eDNA shedding and decay processes, highlighting the importance of considering both biological and environmental factors in eDNA-based monitoring. The findings of this study provide a critical foundation for optimizing eDNA detection strategies for freshwater turtles, particularly in brackish ecosystems subject to salinity fluctuations. • The slider eDNA shedding rate ranged from 10 4 to 10 6 copies/h, with a decay rate of 10 -3 –10 -2 /h. • High salinity significantly decelerated the decay of slider eDNA and promoted its persistence. • Slider density was notably positively correlated with eDNA shedding rate at low salinities (0‰–5‰). • A strong interaction was observed between salinity and density affecting eDNA shedding and decay.