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.