FIGURE 1.

Experimental split‐brood design. From the start of the experiment, clutches of wild winter moth eggs were kept in climate cabinets at a constant 10°C in constant darkness (black timeline). Each clutch was divided into eight subclutches of at least 25 eggs (points). Before the start of the temperature treatment, one subclutch was sampled for RNA extraction (orange arrow at 0 h) and one subclutch was fixed with 4% formaldehyde to determine the median development stage of the clutch with fluorescence microscopy (grey arrow at 0 h). The remaining six subclutches were divided over three temperature treatments (blue arrows), with two transferred to a warm treatment (red line, 15°C), two to a cold treatment (blue line, 5°C) and two remained at baseline temperature (black line, 10°C). For each temperature treatment, we sampled one of the subclutches at 3 h and the other subclutch at 24 h after the transfer for RNA extraction. In total, we gave eggs a temperature treatment in seven development weeks: Every week from 2 weeks after the start of the experiment to week8, six clutches were given a temperature treatment per development week. For developmental weeks 2, 4, 6 and 8, three of these six clutches were used for RNA sequencing, to obtain three biological replicates per sampling point