Comparative study on nuclear and mitochondrial DNA of Ayu Plecoglossus altivelis for environmental DNA-based spawning evaluation

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.