Discordant timing between antennae disrupts sun compass orientation in migratory monarch butterflies

Abstract

To navigate during their long-distance migration, monarch butterflies (Danaus plexippus) use a time-compensated sun compass. The sun compass timing elements reside in light-entrained circadian clocks in the antennae. Here we show that either antenna is sufficient for proper time compensation. However, migrants with either antenna painted black (to block light entrainment) and the other painted clear (to permit light entrainment) display disoriented group flight. Remarkably, when the black-painted antenna is removed, re-flown migrants with a single, clear-painted antenna exhibit proper orientation behaviour. Molecular correlates of clock function reveal that period and timeless expression is highly rhythmic in brains and clear-painted antennae, while rhythmic clock gene expression is disrupted in black-painted antennae. Our work shows that clock outputs from each antenna are processed and integrated together in the monarch time-compensated sun compass circuit. This dual timing system is a novel example of the regulation of a brain-driven behaviour by paired organs.

Figure 1. A single antenna is sufficient for proper time-compensated sun compass orientation

(a) Flight orientation of migrants with only a single antenna and housed in LD (lights on between 0600–1800 hours EST). Migrants were flown between 1300–1500 hours from 29 September to 23 October 2010. The large circle represents the 360° of possible directions with 0° = north. The small solid circles on the perimeter represent the flight orientation of individual butterflies with blue dots representing those with a right antenna and red dots those with a left antenna. Arrow indicates the mean vector and the length of this arrow represents its r value. Left, experimental condition for (A) and (B). (b) Flight orientation of migrants with only a single antenna and housed in a 6-hour-delayed LD cycle (light on between 1200–2400 hours). Migrants with both antennae intact were also tested in the 6-hour-delayed LD cycle (green dots).

Figure 2. Conflicting circadian timing between the two antennae disrupts sun compass orientation

(a) Flight orientation of migrants with a clear-painted and a black-painted antenna. Blue dots, left antenna painted black; red dots, right antenna painted black. Migrants were flown between 1300–1500 hours from 23 October to 20 November 2010. Left upper, experimental condition. (b) Flight orientation of migrants with painted antennae flown a second time after their black-painted antenna was removed. Open dots, first flight (F1 arrow) with clear- and black-painted antennae; black dots, second flight (F2) after black-painted antenna removed. Left lower, experimental condition for (B) and (C). (c) Combined flight light orientation of migrants with only a clear-painted antenna. Green dots, first flight of migrants who had their black-painted antenna removed prior to their first flight trial; black dots, replotted from (B).

Figure 3. Molecular correlates of circadian timing in the antennae and brain

(a) Antenna and brain profiles of per and tim expression in migrants with a clear and black painted antenna. Top, experimental condition for collections. Values are relative to the minimal level for each gene and are mean ± SEM of three antennae or three brains. Points at ZT0 are replotted at ZT24 to show the 24-hr trend. Horizontal bars show the light cycle: open, lights on; grey, lights on in black-painted antenna; black, lights off. P-values, one-way analysis of variance. (b) Antenna and brain profiles of per and tim expression in migrants in which the black-painted antenna was removed. Top, experimental condition for collections.

Figure 4. Clock gene expression patterns do not vary between the antennal bulb and flagellum shaft

(a) mRNA expression in bulb and flagellum. Top inset is schematic of difference in cuticular pigmentation in bulb and flagellum; angled line, the dissection point to separate the two parts of the antenna. (b) Temporal profiles of clock gene expression in bulb (upper) and flagellum (lower) over the course of the day in LD. Values are relative to the minimal level for each gene and are the mean ± SEM of three animals. Statistical analysis performed using one-way analysis of variance. P-values for bulb: cry2, <0.0005; tim,<0.005; per, <0.05; cry1, >0.05. For flagellum: cry2, <0.0005; tim, <0.001; per, <0.01; cry1, >0.05.