Migrant monarchs can use a magnetic field for orientation

We performed indoor magnetic compass trials during the 2012 and 2013 fall migration seasons, in which individual monarchs collected from at least three different locations (see Methods) were tested in a flight simulator that was surrounded by a magnetic coil system used to vary the three different magnetic field parameters (horizontal, vertical, and intensity) (Fig. 1a). To facilitate flight in trials, they were conducted under diffuse white light conditions (spectrum: peak at 600 nm, range: 350–800 nm; total irradiance: 7.45 × 1015 photons s−1 cm−2; Fig. 1b), which also provided migrants with wavelengths of light that are crucial for a functioning magnetic sense in other insects12,13.

Figure 1: Fall migrant monarch butterflies can use a magnetic field to orient in the proper southerly fall migratory direction. (a) Flight simulator and coil system used in magnetic compass trials. (b) Irradiance curve of lighting conditions during flight simulator trials. Light measurement was taken inside the flight simulator, with the flight encoder and diffuser in position, at the position of the head of the tethered monarch butterfly during trials. (c) Orientation of individual directional fall monarchs during the fall 2012 (n=18) and 2013 (n=27) migration seasons (red dots; both years pooled: n=45) in a generated field with a 45° inclination angle, at a 141 μT field intensity. For the circle diagram, arrow indicates mean group orientation, shaded area is 95% confidence interval, mN is magnetic north. Full size image

Migrants from the 2012 and 2013 migratory seasons that were flown under artificial magnetic field conditions (inclination angle=45°; total magnetic field strength=141 μT) and that flew continuously for 5 min were significantly directional with a mean equatorward orientation (2012: α=162°, r=0.707, P=0.0001, n=18; 2013: α=179°, r=0.668, P=0.0001, n=27). There was no difference in orientation direction between years (Watson’s U2 18, 27 =0.028, P>0.5), and the pooled orientation bearing was 172° (r=0.676, P=0.0001, n=45) (Fig. 1c). The 5-min flight duration was previously found to be the minimal time required to evaluate directional orientation behaviour in individual butterflies in the flight simulator6.

For assessing directionality in individual monarchs, we used a Z-value of ⩾500, as in previous publications9,10,14,15 (see Methods). Z, which is the critical value of the Rayleigh test, is calculated by the equation: Z=nr2, in which n is the number of observations and r is the magnitude of the mean vector. Previous studies have shown, using reconstructed flight paths, that directional flight was apparent at Z⩾500 and ambiguous at Z<500 (ref. 14); this Z-value parameter helped define the non-directionality of non-migrant, summer butterflies tested in the flight simulator outdoors under sunny skies.14 Using the Z-value as a measure of directionality, we found that 87% of the 45 monarchs that flew continuously for 5 min exhibited a Z-value of >500. Accordingly, these 39 migrants provide a valid representation of the population of butterflies that flew. They were thus re-flown following the various trial perturbations outlined below with their first flight orientation values used as a control for comparison with subsequent experimental flight studies.

Migrants have an inclination magnetic compass

Most long-distance migratory animals studied to date use the inclination angle component of the Earth’s magnetic field to guide their latitudinal movement in either a poleward or equatorward direction16,17,18. We examined whether or not monarchs also have an inclination magnetic compass by testing the orientation behaviour of individuals when the vertical component of the field was inverted (−45°). This field manipulation alters the directional information provided by the inclination angle such that individuals would perceive it as a 180° shift16,19 and is the acid test for the existence of an inclination compass16,19,20. Eight migrants (initial Z-values: 1,151.0±134.3, mean±s.e.m.; Fig. 2a, left) re-flew for 5 min a second time under the same magnetic field conditions but with the inverted vertical component. The eight migrants re-flown continued to exhibit Z-values >500 (980.7±150.1) but displayed a grouped orientation towards magnetic North (α=1°, r=0.817, P=0.002, n=8; Fig. 2a, right) that was opposite (Moore’s test: R′=1.418, P<0.001) from their initial group orientation (α=175°, r=0.857, P=0.0008, n=8; Fig. 2a, left) by ~

180°. These results demonstrate that individual fall migrants use the inclination angle of a magnetic field as a directional cue, suggesting that monarchs, like birds and sea turtles16,17,18, rely on an inclination compass for long-distance navigation.

Figure 2: The inclination magnetic compass of fall migratory monarch butterflies. (a) Fall migrant monarchs that oriented equatorwards during their initial flight (left; red; n=8), shifted their orientation towards magnetic North when tested a second time under similar generated field conditions (right; dark green; n=8), but with the inclination angle reversed (−45°). (b) Fall migrant monarchs that oriented equatorwards during their initial flight (left; red; n=7), each displayed flight with significantly reduced directionality (that is, Z score <500) when tested a second time under a horizontal field (0° inclination angle) with the same field intensity (n=7). For both a and b, bar graphs indicate the mean Z±s.e.m. score of animals for each group. Circular diagrams are the group orientation behaviour for each group in which each monarch plotted (dot) had a Z score >500. For each circular diagram, the arrow indicates mean group orientation, shaded area is 95% confidence interval, and mN is magnetic north. Full size image

Some animals use a polar compass for directionality and do not reverse their direction when the vertical component is inverted21,22. To rule out a potential contribution of a polar compass to directionality in migratory monarchs, we re-flew migrants at vertical field parameters mimicking those at the equator in which the inclination is 0° but still at a field strength of 141 μT; if the monarch’s magnetic compass is strictly inclinational, then directionality should be severely compromised with only the horizontal (polar) component present (that is, without a vertical component). The seven migrants that re-flew for 5 min under these conditions (initial Z-values 1,087.4±308.4 and group orientation of 146°, r=0.717, P=0.021, Fig. 2b, left) now exhibited a low degree of directionality, flying in circles, with Z scores <500 (69.1±30.9) (Fig. 2b, right). Thus, the inclination magnetic compass is the dominant magnetic compass in monarchs, and the low Z scores represent the default state of orientation behaviour without the use of any inclination information.

The inclination compass uses ultraviolet-A/blue light

We next examined both the light sensitivity and spectral requirement of the inclination response. In migratory birds, the inclination compass is light-sensitive and thought to depend on radical pair-based chemical processes mediated by the flavoprotein cryptochrome (CRY)16,18,23. Given the ability of the two types of monarch butterfly CRYs to restore a light-dependent magnetic response in CRY-deficient Drosophila12,13, it is likely that monarchs also possess a light-dependent inclination compass. This was examined by using long-wavelength pass filters that transmitted wavelengths of light at >420 nm or >380 nm (Fig. 3a). Light intensity through the >420 nm filter was 6.76 × 1015 photons s−1 cm−2 (91% of full-spectrum irradiance), while light intensity through the >380 nm filter was 6.82 × 1015 photons s−1 cm−2 (92% of full-spectrum irradiance). These filters were used because the previous light dependence of the monarch CRY proteins for rescuing magnetosensitivity in CRY-deficient Drosophila was between 380 and 420 nm (ref. 13). When monarchs were exposed to light using the >420 nm filter, the five monarchs that flew for 5 min (initial Z values 985.2±150.1 and group orientation of 162°, r=0.866, P=0.014, Fig. 3b, left) each exhibited a low degree of directionality, flying in circles, with Z–values <500 (9.0±2.6) (Fig. 3b, right); those orientation values were no different from those from migrants without a vertical component (t 6 =1.6023, P=0.1602). When the >380 nm pass filter was used, allowing the addition of light between 380 nm and 420 nm, the five monarchs that flew for 5 min (initial Z-values 1,252.6±406.5 and group orientation of 202°, r=0.92, P=0.006, Fig. 3c, left) exhibited clear directionality with Z-values>500 (658.1±47.8 and group orientation of 157°, r=0.946, P=0.004, Fig. 3c, right). Thus, the monarch inclination compass appears to be light dependent with light in the ultraviolet-A/blue light spectral range (380 nm and 420 nm) important for inclination compass function. The >420 nm and >380 nm filters transmitted light intensities that were 91 and 92% of full-spectrum irradiance, respectively, yet each filter gave strikingly different orientation values. Hence, the contribution of decreased irradiance of the filters to the biological responses was negligible. The importance of light in the spectral range of 380–420 nm for compass function in monarchs is consistent with the inclination response being CRY dependent13.

Figure 3: The inclination magnetic compass uses ultraviolet-A/blue light. (a) Irradiance curves of lighting conditions during flight simulator trials. Black line indicates full spectrum light, blue line denotes filtered light that provided wavelengths of light >420 nm only, and purple line designates filtered light where only wavelengths of light >380 nm were available. (b) Migrants that oriented equatorwards during their first flight when tested under full spectrum lighting conditions (left; red; n=5) had significantly less directional flight, that is, Z<500 (right; blue; n=5), when tested under light that contained wavelengths >420 nm. (c) In contrast, monarchs that oriented equatorwards under full spectrum light during their initial flight (left; red; n=5), maintained similar oriented flight that was equatorwards when tested under lighting conditions that included wavelengths >380 nm (right; purple; n=5). For all trials, the inclination angle was 45° and the field intensity was 141 μT. In both b and c, bar graphs indicate the mean Z±s.e.m. score of animals for each group. Circular diagrams consist of the group orientation behaviour for each group in which each monarch plotted (dot) had a Z score >500. In each circular diagram, arrow indicates mean group orientation, shaded area is 95% confidence interval, and mN is magnetic north. Full size image

The antennae likely contain relevant magnetosensors

We next addressed the location of the magnetosensor. Previous work has demonstrated that the antennae of migratory monarchs are multimodal sensory organs that play an integral role for proper light-dependent, directional flight3,9,10,15. Accordingly, it is possible that the antennae also house the magnetoreceptors necessary for migratory monarchs to utilize the inclination angle of the Earth’s magnetic field. We thus re-tested first fliers under the inverted inclination angle conditions (−45°), but in which re-flown monarchs either had black-painted or clear-painted antennae for the second flight trial. The test condition was evaluated under the inverted inclination angle to rigorously examine the antennae as the potential source of the magnetosensor. Monarchs with black-painted antennae exhibited a low degree of directionality and flew largely in circles during this second flight (Z=127.6±47.6; n=5) (Fig. 4a, right) compared with the directionality of their first flights (Z=1,231.3±181.2 and group orientation of 146°, r=0.826, P=0.024; Fig. 4a, left). The orientation values from migrants with black-painted antennae were no different from those from migrants without a vertical component (t 10 =1.0818, P=0.3047). In contrast, monarchs with clear-painted antennae maintained Z-values >500 (clear-painted: 1,543.8±86.6, n=5 versus unpainted: 1,022.0±193.6, n=5) and were significantly oriented as a group towards magnetic North (α=345°, r=0.9, P=0.009, n=5; Fig. 4b, right) in a direction close to opposite (Moore’s test: R′=1.332, P<0.001) from their initial group orientation in the direction of magnetic South (α=209°, r=0.92, P=0.006, n=5; Fig. 4b, left). Remarkably, these data are consistent with the hypothesis that fall monarchs use a light-dependent, antenna-based inclination magnetic compass for directionality.

Figure 4: The light-dependent magnetosensor involves the antennae. (a) Fall migrants that were oriented equatorwards during their initial flight (left; red; n=5) had significantly less directional flight (that is, Z<500) when both antennae were painted black during their second flight (right; black; n=5). (b) In contrast, fall migrants that were directed towards magnetic South during their initial flight (left; red; n=5) shifted towards magnetic North when tested with clear-painted antennae under reversed inclination angle conditions (right; grey; n=5). For both a and b, monarchs (left diagrams; red) were tested in a generated field with a 45° inclination angle and an intensity of 141 μT. In contrast, monarchs (right diagrams; black for a and grey for b) were tested in a generated field that had the same intensity, but where the inclination angle was −45°. In both a and b, each bar graph indicates the mean Z±s.e.m. score of animals for each group. Each circular diagram consists of the group orientation behaviour for each group in which each monarch plotted (dot) had a Z score >500. For each circular diagram, the arrow indicates mean group orientation, shaded area is 95% confidence interval, and mN is magnetic north. Full size image

The black paint itself was not the cause of the low degree of directionality (Z-values <500) because previous outdoor studies of the time-compensated sun compass have shown that monarchs with black-painted antennae are each highly oriented (Z-values >500), indicating that the sun compass has integrated skylight information (sensed though the eyes) for directionality9. However, because of the desychronized antennal clocks, the result of painting the antennae black, those migrants can no longer orient as a group in the proper flight direction. Intriguingly, without light input to the antennae and directional light cues for retinal processing, the migrants in the present study fly in circles, unable to find any directional cues, as light cues for both the sun compass and inclination compass are absent. In fact, this explains the low degree of directionality and circular orientation patterns that have been observed in another monarch flight simulator study in which individuals were exposed to the >420 nm long-wavelength filter outdoors with only blue sky visible24.

The monarch inclination compass at Earth-strength fields

Although exposure to a strong magnetic field can disorient migrant monarchs thereby suggesting that monarchs may possess a magnetic sense25,26, and even though animals that use a magnetic field for orientation can orient using a field that is considerably stronger than the Earth’s geomagnetic field27, we re-tested first fliers at a field intensity that fell within the range of the Earth’s geomagnetic field (25–65 μT) to determine if the inclination compass of migrants also functions at an Earth-strength field intensity. All re-flown migrants had Z scores >500 (Fig. 5a, right; Fig. 5b). We found that migrants that were oriented equatorward in trials at our stronger field intensity of 141 μT (α=185°, r=0.681, P=0.033, n=7; Fig. 5a, left) were also similarly oriented equatorward (Moore’s test: R′=0.474, P>0.5) when tested in a second flight trial occurring in a magnetic field with a 60° inclination angle, at an Earth-strength field intensity of 57 μT (α=173°, r=0.869, P=0.002, n=7; Fig. 5a, right). Furthermore, consistent with our results that tested migrants under reversed inclination angle conditions at 141 μT (Figs 2a and 4b), migrants that then flew for a third time in trials when tested at 57 μT but with a reversed inclination angle (−60°) switched their orientation behaviour towards magnetic North in a direction near opposite of their second (control) flight (α=331°, r=0.892, P=0.01, n=5; Fig. 5b, right), in which the inclination angle was 60°. Moreover, the orientations of the second control flight (57 μT; 60° inclination angle) and third flight of migrants (57 μT; −60° inclination angle) were significantly different from each other (Moore’s test: R′=1.279, P<0.005). Taken together, these data demonstrate the ability of migrant monarchs to use an inclination compass to orient in the proper equatorward migratory direction, which includes conditions at an Earth-strength field intensity and at an inclination angle encountered during their journey south. The data support the idea that monarchs can use an inclination compass during their actual fall migration in the wild.