2.3 | Sample collection and DNA extraction
Fecal samples were collected by skin diving. We dived underwater with small action cameras (Hero3, Hero 7 white, Hero 7 black, GoPro Inc, San Mateo, Canada) and polyethylene small container (polyethylene petit tube, Ryohin Keikaku, Tokyo, Japan). We recorded individuals that were close to the camera. When the filmed individuals defecated, polyethylene small containers were used to suction the fecal samples along with the environmental water. The recordings were then used to identify which fecal samples were collected from the individuals that defecated. The collected fecal samples were refrigerated in a cooler box on the boat and upon return to land, the samples were processed by discarding the supernatant of the environmental water. The remaining material was preserved at 4 °C after being transferred into containers with 70% ethanol or higher. DNA was extracted from weighing 0.6 g of ethanol-preserved fecal samples using QIAmp DNA Stool Kit or QIAmp Fast DNA Stool Mini Kit (QIAGEN, GmbH, Hilden, Germany) with 200 µl of water. The concentration of extracted DNA was measured with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).
We used a total of 61 fecal samples obtained from 45 individuals between 2014–2021. Due to the nature of fecal samples, some samples failed to quantify DNA methylation rates due to the low concentration of target DNA. Samples in which methylation rates could not be quantified more than twice for each gene were excluded from the analysis. If multiple samples were collected from the same individual but in different years, we treated each sample as an independent dataset. Under this condition, 36 datasets from 30 individuals were used in the analysis. Fig.1 shows the age distribution of the dataset which covers 1–27 years of age.