Understandings of fish spawning are important for the effective riverine ecosystem and fisheries resources management. Application of environmental DNA (eDNA) analysis would allow fish spawning evaluation at a high temporal and spatial resolution due to its less-labor intensive property, compared to the conventional collection methods. However, the relative performance of the two eDNA-based methods used to evaluate fish spawning, pre and post comparisons of eDNA concentrations and nuclear DNA to mitochondrial DNA ratio, has not been compared in the field. We evaluated whether spawning events of Ayu Plecoglossus altivelis could be identified from day (pre-spawning) to night (post-spawning) changes in eDNA concentrations using nuclear DNA (RAG1, nuDNA) and nuDNA/mitochondrial DNA ratio in comparison to the conventional mitochondrial DNA (cyt b, mtDNA). In addition, the relative sensitivity of nuDNA concentrations in spawning evaluation was compared with mtDNA concentrations. Water samples were collected biweekly in the lower Takatsu River, western Japan, between early October and early December 2020, covering the spawning season of the fish species. Generalized linear mixed-effects models (GLMM) showed that Ayu spawning could be detected by nuDNA concentrations likewise by mtDNA concentrations, while nuDNA/mtDNA ratio could not detect spawning earlier in the spawning season. GLS models showed that the day-to-night increase in the eDNA concentrations was more profound for nuDNA than for mtDNA during the main spawning period of the fish. Our findings insist that the application of nuDNA concentrations better serves as a tool for evaluating Ayu spawning intensity in eDNA analysis.

了解鱼类产卵对于有效的河流生态系统和渔业资源管理非常重要。与传统的收集方法相比，环境 DNA (eDNA) 分析的应用将允许在高时间和空间分辨率下进行鱼类产卵评估，因为它的劳动强度较低。然而，用于评估鱼类产卵的两种基于 eDNA 的方法的相对性能，即 eDNA 浓度和核 DNA 与线粒体 DNA 比率的前后比较，尚未在该领域进行比较。我们评估了 Ayu Plecoglossus altivelis的产卵事件是否与传统的线粒体 DNA（cyt b，线粒体DNA）。此外，将 nuDNA 浓度在产卵评估中的相对敏感性与 mtDNA 浓度进行了比较。在 2020 年 10 月上旬至 2020 年 12 月上旬期间，每两周在日本西部的高津川下游采集一次水样，涵盖鱼类的产卵季节。广义线性混合效应模型 (GLMM) 表明，Ayu 产卵可以通过 nuDNA 浓度检测，同样也可以通过 mtDNA 浓度检测，而 nuDNA/mtDNA 比率无法在产卵季节早期检测产卵。GLS 模型显示，在鱼的主要产卵期，nuDNA 的 eDNA 浓度的日夜增加比 mtDNA 更显着。我们的研究结果坚持认为，应用 nuDNA 浓度可以更好地作为评估 eDNA 分析中 Ayu 产卵强度的工具。