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.