Do monarch butterflies use polarized skylight for migratory orientation?
1 VW Nachwuchsgruppe `Animal Navigation', IBU, University of Oldenburg,
D-26111 Oldenburg, Germany
2 Department of Psychology, Queen's University, Kingston, ON, Canada, K7L
3N6
3 Zoological Institute, University of Zürich, Winterthurerstrasse 190,
CH-8057 Zürich, Switzerland
* Author for correspondence (e-mail: henrik.mouritsen{at}uni-oldenburg.de)
Accepted 21 March 2005
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Summary |
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Key words: navigation, time-compensated sun compass, polarized skylight, dorsal rim area, Danaus plexippus, Lepidoptera
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Introduction |
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Polarization vision is a widespread sensory ability of insects
(Waterman, 1981; Wehner,
1982
,
1994
;
Labhart and Meyer, 1999
;
Horvath and Varju, 2004
), and
the pattern of polarized light in the sky is involved in several spatial
orientation mechanisms (Wehner,
1984
,
2001
) ranging from course
control, as suggested for instance in flies
(Wolf et al., 1980
;
von Philipsborn and Labhart,
1990
), to the polarization compass of bees and ants (Wehner,
1984
,
1994
,
1996
). In all insects
investigated so far (bees, ants, flies, crickets and locusts), the ability to
use polarized skylight for orientation is mediated by a group of specialized
ommatidia located at the dorsal margin of the compound eye, termed the dorsal
rim area (DRA) (Wehner, 1982
;
Wehner and Strasser, 1985
;
Fent, 1985
;
von Philipsborn and Labhart,
1990
; Brunner and Labhart,
1987
; Labhart,
1999
; Mappes and Homberg,
2004
). Histological studies
(Labhart and Baumann, 2003
;
Reppert et al., 2004
) and
electrophysiological recordings (J.S. and T.L., unpublished) demonstrated the
presence of a specialized DRA in the monarch butterfly eye. Thus, it was
suggested that monarchs use polarization vision for spatial orientation
(Reppert et al., 2004
). The
aim of the present study is to investigate the role of skylight polarization
in the orientation system of monarch butterflies.
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Materials and methods |
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Experiments were performed outdoors in an open field near Kingston, Canada
(76°30' W, 44°20' N) during the peak of autumn migration
(11 September 26 October). All procedures were approved by Queen's
University Animal Care Committee as compliant with the Canadian Council of
Animal Care Guidelines. Only butterflies flying actively for at least 15 min
were included in the data analysis. For each individual flight, we calculated
the geographical mean flight direction (geo) of the
individual animal and the directedness of its flight,
rindividual (mean orientation vector length;
Batschelet, 1981
). Flights with
rindividual values of <0.1 (40 flights from a total of
over 300) were excluded from statistical analysis, since these flight paths
were undirected. Within each experimental condition, any given individual
monarch was only tested once. To minimize animal use, most butterflies were
tested in several experimental conditions.
To investigate whether migratory monarch butterflies can use the natural
skylight polarization pattern as an orientation cue, we recorded the flight
direction of butterflies under a milky-white, non-UV-transmitting, translucent
Plexiglas lid with a circular opening of 15 cm diameter in the zenith. This
limited the butterflies' view of the natural sky to a 44° visual angle
centered on the zenith. Thus, the butterflies were prevented from seeing the
sun, and the availability of other possible sources of directional information
such as spectral and light intensity gradients was minimized
(Rossel and Wehner, 1984a;
Wehner and Strasser, 1985
;
Wehner, 1997
). Wooden sun
shades erected outside the butterflies' field of view shadowed the simulators
completely, thereby removing potential sun-related directional brightness cues
on the barrel walls. Since maximum skylight polarization occurs at a 90°
angular distance from the sun, the degree of polarization in the zenith is
highest when the sun is near the horizon. Thus, to ensure that the
polarization cues presented were above the perception threshold known from
other insect species (Wehner,
1991
; Labhart,
1996
), we restricted the 44° visual angle experiments to the
morning and afternoon hours when the sun's elevation was less than 40°. In
most of these tests, the sky was completely clear during the entire testing
period. Only in a few tests, up to
20% transient cloud cover was present.
As controls, we tested monarchs with a 120° field of view of clear sky
with the sun visible, and under simulated complete overcast (milky-white
translucent Plexiglas lid without opening) such that neither a view of the sun
nor the polarization pattern was available.
The predicted group orientation responses of monarch butterflies given a
44° view of the clear natural sky depend on how the butterflies are
expected to utilize polarized light cues for spatial orientation. If polarized
light cues are used as a reference for a time-compensated compass system,
monarchs tested in the course of the day should all orient along the migratory
SWNE axis. The directional distribution should be bimodal due to the
nearly full 180° ambiguity of the polarized stimulus in the zenith.
Alternatively, monarchs could line up in a specific preferred direction
relative to the polarized skylight pattern (e.g. to e-vectors perpendicular to
the body's longitudinal axis, as in the fly Musca domestica; see, for
example, von Philipsborn and Labhart,
1990). Their predicted bimodal orientation will then shift over
the course of the day due to the
15 deg. h1 rotation of
the sun and the associated symmetry axis of the polarization pattern.
To analyze our data for such a line-up response, we corrected each
butterfly's mean geographical flight direction (geo) for the
sun-azimuth averaged over the time of the experiment (
sun),
thereby revealing the monarch's orientation (
rel) relative
to the sun azimuth and thus also relative to the perpendicular sun-derived
axis of polarization at the zenith
[
rel=
geo
sun (mod
360°)]. If the monarchs all prefer the same e-vector orientation,
rel is constant. If, however, individual monarch butterflies
have different preferred e-vector orientations, as observed for example in the
locust Schistocerca gregaria
(Mappes and Homberg, 2004
),
the orientation within each experimental group of monarchs will be random.
In a second series of experiments we therefore covered the opening in the
Plexiglas lid with a piece of UVA-transmitting linear polarizer (HN42HE; 3M,
Norwood, MA, USA), providing each monarch with a zenith-centered polarized
light stimulus being 44° wide and having a degree of polarization of
80100% (between 300 nm and 700 nm). After 15 min of active flight, the
lid (including the polarizer) was turned clockwise by 90° in order to test
whether a shift in orientation would occur. As a control, we repeated the
experiments after removing the polarizer from the Plexiglas lid such that the
monarchs could see the natural sky through the opening. The clockwise 90°
turn now caused the same lid movement as before, whereas the polarized
stimulus (natural skylight polarization) remained the same. Only data from
monarchs that kept flying for at least 10 min after the turn had occurred were
analyzed for a shift in orientation. We performed the experiments during
midday (solar elevation >40°), when the degree of polarization at the
zenith was low, and aligned the polarizers ±45° relative to the
average sun azimuth during each experimental 30 min flight period. Since the
e-vector orientation in the zenith is perpendicular to the azimuth of the sun
(Strutt, 1871), this
arrangement ensured that the light intensity underneath the filter was the
same for the two polarizer orientations.
To test if the monarchs could use the artificial polarized light stimulus
for time-compensated orientation, for each flight we calculated (1) the mean
geographical flight direction during the first 15 min of flight
(geo), (2) the geographical orientation of one end of the
artificial polarizer axis during that particular experiment
(
pol) and (3) the sun's azimuth averaged over the time of
the experiment (
sun). From these values, we obtained the
time-compensated geographical heading of the butterfly,
(
comp), as:
comp=
geo
pol+
sun±90°
(mod. 360°). The ±-term in this formula is necessary because any
orientation of the linear polarizer can indicate two equally likely sun
azimuth positions. To allow for this ambiguity, we doubled the angles of the
obtained data, transforming the expected bimodal group orientation into a
unimodal one (Batschelet,
1981
). The value
geo
pol
(mod 360°) indicates the monarch's orientation relative to the polarizer
and is therefore used to test whether the monarchs lined up with the
polarizer.
While we were in the process of writing this paper, Reppert et al.
(2004) reported strong
reactions of monarchs towards an artificial polarizer covering
80° of
the animals' visual field of view. These data contradict our findings using a
44°-wide polarized light stimuli. Therefore, we decided to investigate, in
a third series of experiments performed in autumn 2004, whether the different
sizes of the polarized light stimuli could explain the opposing results. We
mounted a UVA-transmitting linear polarizer (3M® Vikuiti® Polarizing
Film HNP'B; 3M Canada, London, Ontario, Canada; 10.2 cm diameter) 5.7 cm above
the butterflies' heads so as to obtain an 85° highly polarized light
stimulus (degree of polarization >99% between 300 nm and 900 nm): a 0.5
cm-diameter hole was punched into the center of each polarizer, which was then
glued by a small ring of double-sided adhesive tape to the central 1 cm of the
bottom side of the clear, UV-transmitting Plexiglas holder (2 cm diameter), so
that the hole in the polarizer fitted an equivalent hole in the center of the
Plexiglas disc. The disc with the mounted polarizer was fixed on an aluminum
tube (0.5 cm diameter) by using a small fastening screw, so that the position
of the polarizer could be adjusted in height after the 0.5 cm-wide aluminum
tube was slid on the aluminum tube guiding the tungsten rod to which the
butterfly was fastened. We also screwed a clear, UV-transmitting Plexiglas
ring (1 cm diameter) to the bottom end of the guiding tube to prevent the
tightly fitting aluminum tube (and therefore the polarizer) from sliding out
of position. Another UV-transmitting Plexiglas disc (3 cm diameter) was fixed
to the upper end of the aluminum tube just underneath the Plexiglas bar on
which the optical encoder was mounted. This allowed us by turning this disc to
shift the polarizer manually without reaching inside the flight simulator. The
complete HNP'B polarizer holder is illustrated in
Fig. 1. A white, translucent
Plexiglas lid with a 30 cm-diameter opening in the center covered the barrel
and provided a 75° visual field of clear blue sky in the zenith above the
polarizer. Following the experimental procedures described by Reppert et al.
(2004
), we performed all
experiments in the morning and afternoon excluding midday [11:00 h to 13:00 h
Eastern Standard Time (EST)]. The polarizers were aligned parallel to the
polarization pattern in the zenith during the first 15 min of flight and then
turned by 90°, so that they were perpendicular to skylight polarization
during the second part of the flight.
|
The experimental set-up described by Reppert et al.
(2004) did not mention any sun
shades. The sun, even though not directly visible to the butterfly inside the
flight simulator, caused an obvious light intensity pattern on the simulator
walls: the simulator side facing the sun was brightly illuminated through the
white, translucent barrel walls, while the opposite side showed a very
distinct oval-shaped bright spot opposite to the sun's azimuth. In order to
eliminate these brightness artefacts, we repeated the experiments while using
sun shades positioned outside the butterflies' view. These control experiments
were performed late in the season (1226 October 2004). As the maximal
sun elevation was less than 35°, the skylight in the zenith was well
polarized throughout the day so that we could skip the midday break. The
results obtained under the 85° polarized light stimulus were analyzed as
described above for the experiments performed with the 44° polarized light
stimulus.
The last experimental series was designed to test whether the perception of polarized skylight is necessary for migratory orientation in monarchs. Therefore, we painted over the polarized light detectors of clockshifted and non-clockshifted monarchs with opaque black paint [1:1 Lascaux Aquacryl (Alois K. Diethelm AG, Brüttisellen, Switzerland): Marabu Dekorlack (Marabuwerke, Tamm, Germany)], covering the margin of the entire eye except the caudal-most side. The monarchs were tested under a 120° visual field of clear sky including the direct view of the sun. The results were compared with clockshifted and non-clockshifted controls, which did not have their DRA covered. To confirm that the DRA was completely covered by the paint in the experimentals, in 2003 the butterflies were sacrificed immediately after the test flight. In the lab, each eye was mounted individually and sputtered (BAL-TEC SCD 005 Cool Sputter Coater; Balzers, Liechtenstein) with gold (15 nm layer). Then, the paint was peeled off and the probe was sputtered again (10 nm layer). Due to the difference in gold layer thickness, scanning electron microscopy (SEM; S-3200N; Hitachi, Tokyo, Japan) revealed the exact number of occluded ommatidia. The SEM images confirmed that the DRA was amply covered in all animals. Hence, in 2004, we instead peeled the paint off each eye after the experiment had been performed and verified the number of occluded ommatidia based on the clear negative imprint left by the ommatidia on the inside of the painted mask.
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Results |
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Replacing the natural polarized light stimulus with an artificial polarized
light stimulus led to similar results: the monarchs' group orientation towards
the expected e-vector direction at the time of the experiment
(time-compensated orientation) was random under a 44° UVA-containing
polarized light stimulus (doubled angles:
comp=89°/269°, r=0.23, P=0.13;
Fig. 3A). Seen as a group, the
monarchs also did not show any line-up reaction relative to the e-vector axis
of the 44° polarized light stimulus (doubled angles:
geo
pol=174°/354°,
r=0.20, P=0.17; Fig.
3B). Although a shift in orientation after a 90° clockwise
turn of the polarizer was occasionally observed (see Discussion), no
significant group response to a 90° clockwise turn of the polarizer
occurred, and the mean shift in orientation following the turn did not differ
significantly from 0° (
=348°, r=0.61,
P<0.01, 95% confidence interval=31620°;
Fig. 4A). The changes in mean
direction after the turn compared with those before the turn did not differ
significantly from those observed in control experiments where lids with an
opening exposing clear skies but without polarizer were turned
[Fig. 4C; 95% confidence
interval of the mean direction (
) and directedness of the experimental
group (rgroup) overlap with those of the control
group].
|
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Using a larger polarized light stimulus equivalent to that used by Reppert
et al. (2004) resulted in
similar findings: under a 85° UVA-containing polarized light stimulus, the
orientation of the time-compensated group showed a non-significant tendency to
orient on the northeast/southwest directional axis
(
comp=71°/251°, r=0.26, P=0.06;
Fig. 3C). This tendency,
however, disappeared when sun shades shaded the barrel
(
comp=89°/269°, r=0.14, P=0.56;
Fig. 3E). No specific group
alignment towards the e-vector axis of the 85° polarized light stimulus
was observed, either with (doubled angles:
geo
pol=100°/280°,
r=0.16, P=0.47; Fig.
3F) or without sun shades (doubled angles:
geo
pol=42°/222°,
r=0.25, P=0.11; Fig.
3D). The mean orientation shift in the tracks of the monarchs
tested in both shaded or non-shaded barrels was not different from 0°
(Fig. 4B; without sun shades:
=5°, r=0.57, P<0.001, 95% confidence
interval=32428°; with sun shades:
=14°, r=0.7,
P=0.11, 95% confidence interval=32167°).
Migratory, non-clockshifted monarchs with occluded DRAs showed the typical
southsouthwesterly group mean orientation under a clear sunny sky with
view of the sun (geo=208°, r=0.58,
P<0.001; Fig. 5B).
Neither the group mean orientation (95% confidence intervals overlap) nor the
group directedness (non-parametric bootstrap with 5x10 000 replications:
0.39<rgroup<0.78 and
0.72<rgroup<0.90, respectively) differed
significantly from that observed in untreated control butterflies that could
see the sun and the pattern of polarized skylight
(Fig. 5A).
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Discussion |
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The results of our experiments do not provide any evidence that polarized
light cues are used by migratory monarchs in one way or another: monarchs
showed well-oriented migratory flight behavior when given a 120° view of
blue sky including the sun. But if their visual field was restricted to
44° of blue sky without the sun, centered in the zenith, their group
orientation vanished, suggesting that the butterflies were unable to extract
directional information for their time-compensated compass system from the
natural polarized light pattern. The monarchs also did not show any specific
group alignment relative to the skylight polarization axis, as for instance
found in flies (von Philipsborn and
Labhart, 1990). This is in line with the observation that our
monarchs also were oriented at random in relation to the e-vector axis of
UVA-transmitting polarizers providing 44°- or 85°-wide strongly
polarized light stimuli in the zenith. Only in one situation (85°
polarized light stimulus, no sun shades) did the butterflies show a slight
tendency to orient bimodally in a northeast/southwest direction in a
time-compensated manner. However, this effect disappeared after sun shades
excluded distinct light patterns on the flight simulator walls originating
from the sun and which therefore also moved their position with the sun
azimuth.
|
Our results show that monarch butterflies are able to use a
time-compensated compass to orient in their migratory direction without
perceiving polarized skylight via the DRA, as long as they are given
direct view of the sun. Furthermore, after time-shifting their internal clock
by +6 h, the monarchs' group mean orientation was shifted by the predicted
90° independently of whether or not the DRA was occluded. As there
was no significant difference in individual or group directedness of monarchs
with and without occluded DRAs, polarization vision does not seem to add
significantly to the accuracy of the butterflies' compass system. The slight
tendency to reduced group directedness when the DRA was occluded could be due
to the fact that the overpainted area included several hundred ommatidia
outside the DRA. Hence, we conclude that polarized light input is not
necessary for time-compensated sun compass orientation in migratory monarch
butterflies.
The present study also strongly supports our former suggestion that
monarchs do not use the Earth's magnetic field for orientation
(Etheredge et al., 1999;
Taylor et al., 2000
;
Mouritsen and Frost, 2002
): a
total of 140 butterflies was tested without direct view of the sun but with
access to the undisturbed geomagnetic field
(Fig. 7), and their group
orientation was random (
=19°, r=0.09, P=0.33).
The fact that as many as 140 butterflies not seeing the sun still show random
orientation further strengthens our confidence in our flight simulator
results, since even the slightest systematic artifact would have emerged after
testing such a large number of individuals.
|
In conclusion, our study shows that monarchs can use their time-compensated sun compass to orient in their normal southsouthwesterly migratory direction without relying on polarized light information. In other words, polarized light input is not necessary for a time-compensated celestial compass orientation in migratory monarch butterflies. Our data further suggest that monarch butterflies are unable to make navigational use, during their autumn migration, of either a natural or an artificial polarized light stimulus covering a large (up to 85° wide) zenith-centered part of their visual field. Thus, it seems to be the sun and/or the associated light intensity and spectral gradients rather than the pattern of polarized light in the sky that plays the key role in the monarch's time-compensated sun compass guiding the butterflies on their way to Mexico.
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Acknowledgments |
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