Antennae are necessary for time-compensated sun compass orientation. (A) Flight orientation of intact migrants under different lighting conditions. Butterflies were flown between 1100 and 1500 hours from 24 September to 18 October 2008. The large circle represents the 360° of possible directions (0° = north); small solid circles on the perimeter represent the flight orientation of individual butterflies. The arrow indicates the mean vector; arrow length, r value. Left, orientation data of butterflies in LD. Right, orientation data of butterflies in 6-hour delayed LD. (B) Orientation of antennae-less migrants under the different lighting conditions. (C) Free-flight behavior of intact migrants (left bar) and those without antennae (right bar). (D) Temporal profiles of per and tim mRNA levels in brains of monarchs with antennae (blue) and without antennae (red). Values are mean ± SEM of 3 brains. Points at CT0 are replotted at CT24 to show 24-hour trend. Horizontal bars: open, light; black, darkness. p-values, one-way ANOVA.

Circadian clocks in monarch antennae. (A) Clock gene expression in monarch tissues. Tissues were collected at ZT 6 (white bars) and ZT18 (black bars). Values are normalized to those in the brain at ZT18 and are mean ± SEM of 4 animals. p-values, Student’s t-test: ***p < 0.001; **p < 0.01; *, p < 0.05. (B) Clock gene mRNA profiles in antennae. Values are relative to the minimal level for each gene and are the mean ± SEM of 4 antennae. Points at CT0 are replotted at CT24. Horizontal bars: gray, subjective day; black, subjective night. p-values, one-way ANOVA. (C) Circadian profiles of clock protein abundance in antennae. Top, representative autoradiographs in DD. Arrowhead, CRY2 band; the lower band is non-specific, as shown previously (6). Bottom four graphs, quantification of relative protein levels. Values are normalized to the minimal level of protein expression and are mean ± SEM of 3–4 antennae. p-values, one-way ANOVA. (D) Light-sensitivity of antennae in culture. Top, experimental scheme in LD or in phase-reversed LD (R-LD). Arrows, collection times. Bottom, western blot analyses. Left, representative blots; right, quantifications. Open bars, mid-light; black bars, mid-dark. Values are mean ± SEM of 5 antennae. p-values, Student’s t-test: **, p < 0.01; *, p < 0.05. (E) Daily and circadian patterns of TIM abundance in two sets of cultured antennae (gray and black lines). Top, lighting conditions. Bottom, representative western blot.

Blinding antennal clocks alters their timing. (A) Irradiance curves for different painting conditions. Light measurements were taken under full-spectrum light through plastic that was either painted or not. (B) Light-sensitivity of TIM abundance. Top, experimental paradigm. Painted antennae were harvested at CT18 (arrows). Middle, blot of TIM levels from pooled antennae painted clear (C) or black (B) from butterflies in dark or light pulsed. Bottom, quantifications. Values are mean ± SEM of 3–4 antennae. White bars, clear-painted antennae; black bars, black-painted antennae. p-values, Student’s t-test: **, p < 0.005. (C) Temporal patterns of per and tim expression in brains (left column) and antenna (right column) from butterflies with the antennae painted clear or black. Values are mean ± SEM of 3 animals, except for the three points without error bars that represent the mean of 2 animals. Box shading: open, light; dark gray, night; and light gray, subjective day. p-values, one-way ANOVA.

Blinding antennal clocks alters sun compass orientation. (A) Flight orientation of migrant butterflies with intact antennae (control, upper) and with antennae painted clear (middle) or black (lower). Butterflies were flown between 1100 and 1500 hours from 20 October to 16 November 2008. Left, butterflies housed in LD. Right, butterflies housed in 6-hour delayed LD. (B) Relationship between orientation angle and day of study. Individual orientation directions were standarized to the mean vector of control butterflies (144º = 0º) and assumed to be drifting from the mean in a counterclockwise direction over time. Black dots, LD 0600–1800; open dots, 1200–2400. p-value, linear regression analysis.