Aquarium experiment
The aquarium experiment was performed to examine how the relationships
between fish abundance and target eDNA concentration varied depending on
filter pore size and DNA fragment length. Five replicates of 25-L tanks,
filled with 20 L of aged tap water aerated by a pump, were prepared. The
water temperature was regulated (around 25°C) using a thermostat (DS150,
GEX, Japan), and a 12-h/12-h light/dark cycle was adopted throughout the
experiment. After a week, 500 mL of tank water was collected from each
tank in advance, using a 1-L plastic beaker as a blank sample, and
subsequently, five individuals of the juvenile zebrafish (total length:
around 30 mm; no sex identification) were introduced into each tank.
Then, after 5 days, 3 L of water samples were collected from each tank
using a 10-L plastic container, introducing an additional five juvenile
zebrafish (i.e., ten individuals per tank in total). After another 5
days, we collected 3 L of water samples similarly as above and
introduced an additional ten individuals (i.e., 20 individuals per
tank). Similarly, we collected water samples after 6 days and introduced
an additional ten individuals (i.e., 30 individuals per tank). After 10
days, 3 L of water samples were collected, and ten additional
individuals were introduced (i.e., 40 individuals per tank). Finally,
after 5 days, we collected 3 L of water samples.
After water collection, the water samples were thoroughly mixed and 250
mL of them was filtered using a 47-mm-diameter polycarbonate membrane
filter (0.2 μm pore size; Merck Millipore, Germany) using magnetic
filter funnels (500 mL capacity; Pall Corporation, US) and a filtration
manifold (three-place; Sanplatec Corporation, Japan). A similar
procedure was performed for 500 mL of the samples with the same material
filters with different pore sizes (0.8, 3, and 10 μm pore size). This
alternative was performed as a result of clogging of smaller pore size
filters. Additionally, 250 mL of distilled water was filtered with the
same material filter at each sampling time, with 0.2-µm pore size filter
used as a negative filtration control. All filtered samples were covered
with a commercial aluminum foil and kept at −20°C until eDNA extraction.
The fish in experimental tanks were fed a small amount of commercial
bait every 2 or 3 days. The bottom of each tank was cleaned immediately
after feeding to eliminate the effect of the feces. Tank water was
filled up to the original water volume after water sampling and bottom
cleaning was performed. We supplied replacements for the dead or dying
individuals as soon as possible. However, we replaced them after water
sampling when the dead or dying individuals were observed on the
sampling day. Disposable gloves were worn to collect and filter water
samples throughout the experiments. All the equipment for fish rearing
(pomp, air stones, tube, and acrylic tanks), water collection (beakers,
containers, polyethylene tanks), and water filtration (filter funnels
and tweezers) were bleached before every use in 0.1% sodium
hypochlorite solution for at least 5 min (Yamanaka et al., 2017).