Particle size influences decay rates of environmental DNA in aquatic systems

Environmental DNA (eDNA) analysis is a powerful tool for remote detection of target organisms. However, obtaining quantitative and longitudinal information from eDNA data is challenging, requiring a deep understanding of eDNA ecology. Notably, if the various size components of eDNA decay at different rates, and we can separate them within a sample, their changing proportions could be used to obtain longitudinal dynamics information on species. To test this possibility, we conducted an aquatic mesocosm experiment in which we separated fish-derived eDNA components using sequential filtration to evaluate the decay rate and changing proportion of various eDNA particle sizes over time. We then fit four alternative mathematical decay models to the data, building towards a predictive framework to interpret eDNA data from various particle sizes. We found that medium-sized particles (1-10 μm) decayed more slowly than other size classes (i.e., <1 μm and >10 μm), and thus made up an increasi..., Filter DNA extraction and quantification
DNA was extracted from preserved filters using Phenol-Chloroform extraction (Sambrook & Russel, 2006), following the protocol available in the Supplement, modified from Turner et al., (2014). We resuspended the extracted DNA in 100 μl of TE buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0), and stored it at -20°C until genetic analysis. We included an extraction negative control of RO water for every 24 samples.
We quantified target eDNA using a duplex digital droplet PCR assay (ddPCR) and species-specific primer-probe pairs developed by Takahara et al. (2012) for Common carp (C. carpio) and by Duda et al. (2021) for Rainbow trout (O. mykiss) that both target the mitochondrial gene Cytb. These primers and probes had been previously applied for qPCR only and in separate reactions, so we combined them with distinct labeling to validate a duplex ddPCR assay. The reactions were composed of 11 μl of ddPCR Supermix for probes (Bio-Rad, Inc., Hercules, CA), ...,