Experimental set-up
We tested for effects of fish kairomones, subpopulation and their
interaction on the metabolomes in a full factorial experiment. The total
design consisted of 6 clones × 3 subpopulations × 2 fish kairomone
treatments × 8 replicates = 288 experimental units.
To manipulate fish predation risk, fresh medium containing fish
kairomones was added daily. To prepare the medium, three fish (5-7 cmGasterosteus aculeatus sticklebacks) were kept for 24 h in 20 L
aerated and bio-filtered tap water. This fish-conditioned water was
filtered twice (0.45 µm) and diluted five times to obtain a final
concentration of three fish per 100 L, which is known to generate strong
responses in D. magna (Pauwels et al. 2010). The fish were
fed D. magna daily in a separate bucket to avoid the presence ofDaphnia alarm cues in the fish medium. The culture medium was
refreshed every other day. The medium refreshment was performed between
9:00 and 12:30 and feeding was done at 13:00 during the entire culturing
period to maintain consistent.
To obtain enough synchronized juveniles to start the experiment, for
each clone we cultured ten to twelve Daphnia mothers from one
grandmother. Cohorts of 16-18 juveniles of the pooled second brood of
these mothers, all born within a 24 h interval, were used as
experimental animals and cultured in 500 mL glass vials filled with 450
mL bio-filtered tap water. During the experiment offspring was daily
counted and removed from the vials. The experiment was ended when the
animals had released their second clutch. The Daphnia developed
relatively synchronously so that we could stop vials when all animals
had released their second clutch and while there were no visual signs of
the third clutch in the brood pouches. Animals were checked every 12 h
and were not fed for the last 12 h before sampling to ensure empty guts.
Per vial, we collected three individuals for metabolomic profiling. All
samples were flash frozen in liquid nitrogen and then stored at -80 °C.