Sexually mature fishes were collected in the waters off Zhoushan (30°39 N,
122°11 E), Zhejiang, China. Water of different salinity was prepared starting
from filtered 29‰ seawater in which P. punctatissimus lives, which was then
either diluted with double distilled water to reach salinities below 29‰ or
concentrated seawater prepared with Instant Ocean salt was added to obtain
salinities above 29‰; the natural seawater (29‰) served as the control medium.
Salinity was measured by refractometry.
Because freshly caught pomfrets are difficult to maintain in an experimental
setting, a ‘dry method’ was used to obtain zygotes by in-vitro fertilization. Eggs
were gently squeezed from the gravid female into a beaker immediately after
capturing. A male was then squeezed to release milt directly onto the eggs. Eggs
and milt were gently stirred and well mixed and allowed 2 min for fertilization to
occur. Fertilization was determined based on 100 eggs under a dissecting
microscope. Eggs were then transferred to 1000 ml plastic bottles containing
water of different salinities, ranging from 0‰ to 50‰, at 5‰ intervals. Water
temperature was 26.0–28.0 °C. Each bottle contained about 300 fertilized eggs
(sorted under a dissecting microscope), with 3 replicates for each salinity