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.