To assess circadian rhythmicity, we maintained beetles under 16L:8D conditions for more than 20 days in an incubator kept at 25°C before the measurement of locomotor activity, and we then measured the locomotor activity of T. castaneum for 10 days in darkness. A beetle from each population was placed in a clear plastic Petri dish (30 × 10 mm) in an incubator (MIR-153, Sanyo, Osaka, Japan) maintained at 25°C under complete darkness (DD). We measured the circadian rhythms of 17 to 128 individual beetles for a single population. The sample size of each population measured for circadian rhythm is shown in S1 Table. The locomotor activity of each individual was monitored using an infrared actograph. An infrared light beam was passed through a clear Petri dish, and the beam was projected onto a photomicrosensor (E3S-AT11; Omron, Kyoto, Japan) that detected all interruptions of the light beam. Signals of interruption of the infrared light beam were recorded every 6 min [11]. At a time, we monitored 128 individuals, meaning we are measured 128 sensors at the same time.

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