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), ...,

环境DNA（Environmental DNA, eDNA）分析是一种用于远程检测目标生物的强有力工具。然而，从eDNA数据中获取定量且纵向的信息颇具挑战，这需要对eDNA生态学具备深入认知。值得注意的是，若eDNA的不同尺寸组分具有不同的降解速率，且我们可在样本中对其进行分离，则可通过其比例变化获取物种的纵向动态信息。为验证这一可能性，我们开展了水生中宇宙实验：通过连续过滤分离鱼类来源的eDNA组分，以此评估不同尺寸eDNA颗粒随时间推移的降解速率与比例变化。随后，我们针对实验数据拟合了四种备选的数学降解模型，旨在构建可用于解读不同尺寸颗粒eDNA数据的预测框架。研究发现，中等尺寸颗粒（1~10 μm）的降解速率慢于其他尺寸组别（即<1 μm与>10 μm颗粒），因此其占比呈递增……

### 滤膜DNA提取与定量分析
我们采用酚-氯仿提取法（参考Sambrook与Russel, 2006年的方法）从保存的滤膜中提取DNA，实验流程遵循补充材料中记载的、经Turner等人（2014年）修改后的方案。将提取得到的DNA重悬于100 μl TE缓冲液（10 mM Tris，0.1 mM EDTA，pH 8.0）中，并于-20℃条件下保存，直至开展遗传分析。每24个样本设置1组反渗透（RO）水提取阴性对照。

我们采用双重数字液滴PCR（digital droplet PCR, ddPCR）检测体系，以及针对线粒体基因Cytb设计的物种特异性引物-探针组合，对目标eDNA进行定量：其中鲤（C. carpio）的引物-探针组合由Takahara等人（2012年）开发，虹鳟（O. mykiss）的引物-探针组合由Duda等人（2021年）开发。此前，这些引物与探针仅应用于实时定量PCR（qPCR）且为单独反应体系，因此我们通过设计不同的荧光标记对其进行组合，以验证双重ddPCR检测体系的可行性。反应体系包含11 μl探针用ddPCR预混液（Bio-Rad公司，加利福尼亚州赫拉克勒斯市），……