2.3. Multi-generational experiment and measurement on lifespan and reproduction
In preliminary experiments, DEHTP at 8.0 mg/L provoked lethality smaller than 5% (LC5). Accordingly, the nominal exposure concentrations were arranged as 0.0008, 0.008, 0.08, 0.8, and 8.0 mg/L, three of which represented realistic μg/L levels (Bulbul et al., 2022). Briefly, warm NGM were mixed with DEHTP stock solutions or sterile K-medium solutions containing 1% DMSO (as the control) with a volume ration of 99:1 to reach the nominal concentrations. Then, they were aliquoted into Petri dishes. After cooling down to form agar, they received the same amounts of E. coli OP50 suspensions with the same bacterial density. Then, they were kept overnight for mild evaporation to form a bacterial lawn. At last, the age-synchronization eggs were added on the NGM agars to start the first-generation exposure (marked as F1) (Li et al., 2019; Yu et al., 2017; Yue et al., 2021). Each experimental group including the control had at least ten replicates of NGM agars.
On the 3rd day of the F1 exposure, in each experimental group the nematodes were collected and randomly allocated to four experimental manipulations: (1) 48 adults were transferred onto 48 NGM agars with continuous treatment of DEHTP or K-medium solutions to measure lifespan and reproduction where disappeared nematodes were not calculated (Liu et al., 2020; Yu et al., 2017); (2) approximately 1000 adults were transferred onto clean NGM agars for behavior measurement; (3) 200 adults were transferred onto new NGM agars with continuous DEHTP or K-medium treatment to reproduce eggs for 24 h. Then, the adults were discarded, and the age-synchronized eggs started next generation exposure (i.e., F2) (Yue et al., 2021); (4) the remaining nematodes in each group were collected to 20 centrifuge tubes and stored at -80 ºC until assayed. The procedure was repeated to finish a multi-generational exposure with 4 consecutive generations from F1 to F4.