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.