Pupil dynamics reveal the tuning of tortricid moths to diel activity.

Insect compound eyes adapt in response to average stimulus intensity, but the adaptation is mediated also by intrinsic circadian rhythms and abiotic factors like temperature, which are indirectly related to the stimulus. We studied the effect of light, circadian rhythm, temperature and body size on light adaptation in Grapholita molesta (GM), Lobesia botrana (LB) and Cydia pomonella (CP) (Lepidoptera: Tortricidae). These moths have superposition compound eyes with identical trichromatic photoreceptor sets and temporal resolution; however, the adults are sexually active before (GM), during (LB) or after (CP) sunset, and experience very different light intensity and temperature variations during their activity period. Their eyes were examined with infrared pupillometry, light microscopy and micro-CT. The compound eyes are very small to medium-sized, with a clearly visible superposition pupil. The pupil reflectance of adults entrained to a 12/12 h light/dark cycle for 2 days maintained the entrained rhythm for days, closing and opening during the subjective day and night, respectively. Circadian rhythm was the most robust in CP. A fully open pupil was forced to close with a light pulse, and pupil brightness changes were monitored at 15 and 22 °C. The experiment revealed complex and different pupil dynamics among species at both temperatures. GM, the smallest species, was most affected by the lower temperature. The experimental paradigm can be used for high-throughput, non-invasive monitoring of multiple species' response to climate change, and to chemical and light pollution.