Stability of environmental <scp>DNA</scp> methylation and its utility in tracing spawning in fish

The use of environmental DNA (eDNA) is becoming prevalent as a novel method of ecological monitoring. Although eDNA can provide critical information on the distribution and biomass of particular taxa, the DNA sequences of an organism remain unaltered throughout its existence, which complicates the accurate identification of crucial events, including spawning. Therefore, we examined DNA methylation as a novel source of information from eDNA, considering that the methylation patterns in eggs and sperm released during spawning differ from those of somatic tissues. Despite its potential applications, little is known about eDNA methylation, including its stability and methods for detection and quantification. Therefore, we conducted tank experiments and performed methylation analysis targeting 18S rDNA through bisulphite amplicon sequencing. In the target region, eDNA methylation was not affected by degradation and was equivalent to the methylation rate of genomic DNA from somatic tissues. Unmethylated DNA, abundant in the ovaries, was detected in the eDNA released during fish spawning. These results indicate that eDNA methylation is a stable signal reflecting targeted gene methylation and further demonstrate that germ cell-specific methylation patterns can be used as markers for detecting fish spawning.