Fig. 1: A robust individual-level behavioral assay for light-dependent magnetoreception in the monarch butterfly.

a Monarchs are tethered and fixed in the center of a flight simulator surrounded by a 3D Helmholtz coil system, facing the geomagnetic South. Of the three pairs of coils (red, blue, and green), the plane of the green pair is parallel to geomagnetic North (N) and perpendicular to geomagnetic east (E). b Frontal view of the tethered monarch and the infrared beam system counting the number of wingbeats. c Irradiance curve of full-spectrum light conditions (~350–800 nm) during trials. d Left panels, North-East-down coordinate system of the ambient magnetic inclination (AMI) and the reversal of AMI (RAMI). B_F, B_H, B_Z, and I represent the geomagnetic vector, horizontal component, vertical component, and inclination of the magnetic field, respectively. Right panels, magnetic responses of wild-caught migratory (orange) and laboratory-raised (black) wild-type monarchs to a reversal of magnetic inclination (red line) under full spectrum light (white circle) (n = 32 for each group). Each box plot shows the number of wingbeats for every 10 s time bin (median, centerline; interquartile range (IQR), box; 1.5× IQR, whiskers). Each dot represents the response of an individual. Statistical significance between wild-caught migrants and laboratory-raised monarchs was tested for each time bin using a two-tailed Mann–Whitney U test at p < 0.05. No significance was found.