Experimental design
For the F0 generation, a similar experimental design as
in Issa et al. (2020) was used, corresponding to a full factorial design
with control, dopamine and two food rations (high versus restricted),
with six 500 mL replicate beakers (non-aerated borosilicate beakers,
Fisherbrand) for each of the four combinations (Fig. 1). Ten neonates
(< 24 h old) were introduced into each beaker and kept at 20
°C under long photoperiods (16h L: 8h D) until maturation. The exposure
concentration of dopamine (2.3 mg/L) was chosen for successfully
inducing changes in D. magna growth (Issa et al., 2020; Weiss et
al., 2015). An exposure protocol as previously outlined in Issa et al.
(2020) was followed. Specifically, dopamine hydrochloride
(Sigma-Aldrich, St. Louis, MO, USA) was first dissolved in 100 mL
ultrapure water before dilution in ADaM to the desired exposure
concentration. Controls containing only ADaM medium were performed
parallel to the exposure replicates. The medium was renewed in all
replicates (N = 24) three times a week, and the animals were fed at each
renewal event with Shellfish Diet 1800® at a final concentration of 2.88
× 105 cells/mL (ad lib at 20 °C) for the high
food ration and 8.6 × 104 cells/mL (30% ad libat 20 °C) for the restricted food ration.
All replicates were checked daily for mature individuals (defined as
having their first clutch of eggs released into the brood chamber).
Mature individuals were removed from the beakers, photographed for size
measurements (see section 2.3 below) and placed individually in 2 mL
non-aerated wells containing ADaM at 10 °C to give birth. Seventeen of
the 240 individuals in the beakers were found to be males and were thus
not transferred (Fig. 1). To prevent resulting neonates from obtaining
food upon birth, no food was added to the wells. The wells were checked
daily and once neonates had been released (i.e. < 24 h old,
F1 generation), two individuals were randomly sampled
from each well, photographed for size measurements (see section 2.3
below) and transferred individually to new 2 mL non-aerated wells
containing ADaM (no food) at 10 °C. The medium was not exchanged, and
individuals were checked daily to record mortality. The low exposure
temperature was chosen to slow down metabolism and increase longevity
under starvation, thus making it more likely to accurately quantify any
variation in offspring longevity under the chosen frequency of
observations.