Circadian rhythms, their free-running periods and strength of the rhythm are often used as indicators of biological clocks, and there is evidence that the free-running periods of circadian rhythm are not affected by environmental factors like temperature. However, there are few studies of environmental effects on the power of rhythms and it is not clear if temperature compensation is universal. Additionally, genetic variation and phenotypic plasticity in biological clocks are important for understanding the evolution of biological rhythm, but genetic and plastic effects are rarely investigated. Here, we used 18 isofemale lines (genotypes) of Gnatocerus cornutus to assess rhythms of locomotor activity, while also testing for temperature effects. We found that total activity and power of circadian rhythm were affected by interactions between sex and genotype or sex, genotype and temperature, so that while males tended to be more active and showed greater increases in activity, this effect varied across both genotypes and temperatures. The period of activity only varied by genotype and was thus independent of temperature. The complicated genotype-sex-environment interactions we recorded stress the importance of investigating circadian activity in more integrated ways.

To assess circadian rhythm, we maintained beetles at 14L10D conditions for more than 20 days in an incubator kept at 25^oC before the measurement of locomotor activity, and we then measured the locomotor activity of G. cornutus for 10 days in darkness. A beetle from each isoline with enough food described above was put in a clear plastic Petri dish (30 × 10 mm) in an incubator maintained at 25^oC or 29^oC. The beetles develop faster at 29℃ (unpublished observation, TM), and almost investigation of the beetle’s behaviour has been conducted under 25^oC (Okada et al. 2006; Okada and Miyatake 2009, 2010), and thus we choose the two temperatures, 25^oC and 29^oC in this experiment. 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. Sample size of each isoline is shown in S1 Table.