Neural coding underlying the cue preference for celestial orientation

Nocturnal Dung Beetles Use the Polarization Pattern as a Primary Orientation Cue at Night.

Dung beetles use a large repertoire of celestial cues for orientation (5, 7, 15–17), but their two main compass cues are (i) the sun or moon and (ii) the celestial polarization pattern (5). Here, we first sought to test if one compass cue is dominant over the other in the orientation systems of two closely related dung beetle species that are active at different times of the day: Scarabaeus lamarcki (diurnal) and Scarabaeus satyrus (nocturnal). The robust straight-line orientation behavior of these beetles can be induced independent of the time of day, allowing us to examine the beetles’ choice of celestial cues even at times when they are not usually active (i.e., the nocturnal species during the day, the diurnal species at night). First, we measured the change in the beetles’ directions between two consecutive rolls when they rolled their balls out of an arena (1-m diameter) on a sunlit day or under a clear, moonlit sky (Fig. 1 B and C; control, ●). We tested the beetles’ orientation behavior at low sun elevations or (on full-moon nights) moon elevations (<30°). Under these conditions, a celestial body (sun or moon) and the celestial polarization pattern created by it are both clearly visible to the animals (Fig. 1A). In all cases, the change of direction between the two repetitions was clustered around 0° (P < 0.001 by V test, with an expected mean of 0°); that is, the beetles did not change their bearing between consecutive rolls (Fig. 1 and Figs. S1A and S2 A–D). This observation confirmed that both species have a consistent orientation behavior even at times when they are not naturally active.

To investigate the relative importance of, or preference for, the two main celestial cues in the beetles’ compass system (sun or moon vs. polarized light), we also examined how beetles reacted when the two cues were set in conflict. Again, beetles were made to roll out from the center of the arena on two consecutive occasions: once in the light of the sun or the moon and once with the sun or full moon (celestial body elevations <30°) “displaced” to the opposite sky hemisphere using a mirror (at the start of either the first roll or the second roll), while simultaneously hiding the real sun or moon from the beetle’s view (Fig. 1 B and C; test, ○). If the beetle relies on polarized skylight as its main cue, it should adhere to its original rolling direction, which would still be visible (and unaltered) across the remainder of the visible sky. In contrast, if the beetle uses a celestial body (sun or moon) as its primary reference for orientation, it should use the reflected moon or sun as a directional cue and turn by about 180° with respect to its original rolling direction. We found that the result depended on which species was tested. Diurnal beetles followed the reflected sun or moon (i.e., they changed their rolling direction significantly toward the opposite sky hemisphere), both during the day (P < 0.001 by V test, with an expected mean of 180°) and at night (P = 0.002 by V test, with an expected mean of 180°) (Fig. 1 B and C, Left and Figs. S1A and S2 A and C). This result matches our previous findings (5, 17, 18) and suggests that diurnal dung beetles rely primarily on a celestial body for orientation; that is, they use either the sun or the moon as a compass. This cue preference was also true for the nocturnal species when orienting during the day [Fig. 1B, Right (P < 0.001 by V test, with an expected mean of 180°) and Figs. S1A and S2B]. However, when these nocturnal foragers rolled their balls in the light of the full moon, they displayed a different strategy: The beetles did not change their rolling direction (P = 0.996 by V test, with an expected mean of 180°), but kept rolling in their original direction of travel even when the moon was reflected back at them from the opposite direction [Fig. 1C, Right (P < 0.001 by V test, with an expected mean of 0°) and Figs. S1A and S2D]. In contrast to the diurnal species, the nocturnal species thus primarily followed the direction given by the skylight polarization pattern under dim light conditions. To confirm this observation, we also tested the nocturnal species rolling under a polarizing filter with a clear view of the full moon (elevation <30°) through the polarizer. The polarizer was placed on top of a screened arena, with the filter’s E-vectors either aligned (Fig. S3A) or in conflict (Fig. S3B) with the lunar polarized skylight. In contrast to the diurnal species, which orients with respect to the sun even if the polarizer is turned by 90° (5), the nocturnal species significantly changed its bearing in response to a change in the polarized light direction (Fig. S3). This experiment also shows that polarized light is ranked higher than the disk of the moon in the internal compass of nocturnal dung beetles. Overall, we show that despite the taxonomic proximity of both species, they rely on different celestial cues for their dominant directional reference under dim light conditions. Because both species exhibit similar orientation behaviors but differ in their activity window, our data suggest that nocturnal conditions (presumably the lower light intensity), or a circadian rhythm, cause the observed switch to a polarization compass in the nocturnal species.

Central Complex Cells Are Sensitive to Polarized UV Light.

Next, we sought to understand how the polarized light pattern and the position of the sun or moon are processed neurally in the beetle’s brain. To allow an accurate investigation of the neural substrate for polarization-dependent orientation, we first had to define at which wavelength of light the beetles perceive the skylight polarization pattern. Dacke et al. (15) suggested that dung beetles detect polarized light either in the green or UV wavelength. Therefore, we investigated the orientation performance of both of our model species in an indoor arena lit by polarized light from above, with a peak either in the UV (365 nm) or green (530 nm) wavelength (Fig. 2 A and B and Fig. S4). Each beetle was made to roll a ball twice from the center to the perimeter of a circular indoor arena. Between each roll, the polarizer either remained in place (control) or was turned by 90° (test). Under polarized UV light, both species changed their heading by 90° [Fig. 2B (in both species, P < 0.001 by V test, with an expected mean of 90°) and Figs. S1B and S2 E and F], whereas under polarized green light, the beetles were disoriented (Figs. S1B and S4). Thus, as in many other insects (19–22), both of our dung beetle species detect the skylight polarization pattern in the UV range.

To analyze the tuning of the polarization-sensitive neurons, we presented the beetles with zenithal polarized UV light that had similar light intensities as in the behavioral experiments. Before starting to roll, dung beetles always climb on top of their balls and perform “orientation dances” in which they rotate around their own body axis (23). To simulate this vertical rotation underneath the natural polarization pattern, we rotated the polarizer by 360° while recording from compass neurons of the central complex. The dung beetle’s central complex consists of four brain areas, which have been described in detail in other insects (24): the upper and lower divisions of the central body (termed the fan-shaped and ellipsoid body in flies), the protocerebral bridge, and the paired noduli (Fig. S5 A–C).

Using single-cell tracer injections, we were able to identify (and record from) all four types of central complex neurons known to process polarization signals in other orienting insects (14) (Fig. S5D). According to a proposed processing network in the locust’s central complex (25), these cells can be categorized into early and late processing-stage neurons: Input tangential (TL) neurons and columnar (CL1) neurons represent the early processing stage, whereas tangential (TB1) cells and columnar (CPU1) neurons represent the late processing stage (Fig. S5D). In total, we recorded from 21 central complex neurons [TL: n = 8 (three diurnal, five nocturnal), CL1: n = 9 (four diurnal, five nocturnal); TB1: n = 3 (one diurnal, two nocturnal); CPU1: n = 1 (nocturnal)] while rotating the polarizer by 360° above each beetle at least twice (one clockwise rotation and one counterclockwise rotation) (Dataset S1).

Neurons of the same type showed similar responses, independent of the species’ identity. When stimulating early processing-stage cells (TL and CL1) with a rotating polarized UV light, the neurons were maximally excited at a particular orientation of the polarizer and were maximally inhibited at the perpendicular orientation (Fig. 2 C and D), demonstrating their angular sensitivity to polarized UV light. In contrast, late processing-stage cells (TB1 and CPU1) typically showed a firing activity that was similarly, but less strongly, modulated during polarizer rotation (Fig. 2D and Fig. S6 A and B). This neural tuning is well in line with observations from the locust’s central complex and can most likely be explained by a multimodal function of the late processing cells in the central complex network (14, 25). In summary, the dung beetle’s central complex acts as a key processing center for polarized light analysis. However, to function as a robust internal compass for straight-line orientation, it would also have to identify and integrate the azimuthal position of the sun (or moon), as has recently been shown for locusts and monarch butterflies (26, 27).

Central Complex Neurons also Encode the Sun/Moon Azimuth.

During our experiments, we also analyzed how the very same neurons that responded to polarized light were modulated in response to a moving unpolarized green light stimulus. In honey bees, a green light spot is interpreted as the sun’s direction (28–30). To test whether the sun or moon can be replaced by an artificial green light spot also in the compass system of the beetles, we let the beetles roll first under the clear sky and then again immediately afterward in an indoor arena, lit only by a green light spot (or vice versa, using the light spot first followed by the celestial cue) (Fig. 3 A and B). Interestingly, even when the beetles had a green light spot as their sole orientation cue, their orientation was equally precise (nocturnal species) or only slightly less precise (diurnal species) than their performance outdoors when many celestial cues were available (Fig. S1C). We found that irrespective of whether the beetles (n = 20 diurnal beetles and n = 20 nocturnal beetles) first rolled under the green light spot (elevation about 25°) or under the sun or moon, the beetles’ heading was maintained in both situations (i.e., the change of direction was clustered around 0°) (Fig. 3C; in both species, P < 0.001 by V test, with an expected mean of 0°; Fig. S2 G and H). Both dung beetle species thus clearly orient in a similar way to a green light spot as they do to a celestial disk.

Next, we simulated beetles rotating under the sun or the moon (but in the absence of a celestial polarization pattern) and analyzed the response of 20 neurons to a moving green light spot that moved on a circular path around the beetle’s head. In total, 19 (TL: n = 7, CL1: n = 9, TB1: n = 3) of the polarization-sensitive central complex neurons recorded above showed a significant response also to the moving green light spot (Fig. 3 D and E; P < 0.05 by Rayleigh test) and were clearly modulated by the stimulus position (Fig. S6 A and B). Early processing-stage cells (TL and CL1) were excited by the green light spot when it passed a certain position in the visual field. TL neurons showed an excitation with a narrow receptive field. In contrast, CL1 neurons showed an additional strong inhibition in the opposite visual field (Fig. 3E). Two CL1 neurons showed only inhibition as a response to the green light spot. In contrast, the response characteristics of late processing-stage (TB1) cells were diverse but always featured an increased average neural activity at a certain azimuth of the light spot (Fig. 3E). In summary, our data show that compass neurons in the beetle’s central complex encode the pattern of polarized skylight and are suited to encode the sun’s or moon’s azimuth (especially cells at an early processing stage). In combination, these two cues can be used to generate a robust internal sky compass signal for straight-line orientation. Next, to test the relative representations of both signals at the neural level, we set out to analyze the neural activity of central complex cells when the animal was exposed to both stimuli simultaneously.

Tuning of Central Complex Neurons Matches the Beetles’ Behavioral Cue Preference.

Behaviorally, we found that diurnal dung beetles primarily use a celestial body for orientation, both during the day and at night. Nocturnal dung beetles also use this cue under daylight intensities but switch to a polarization compass for nocturnal orientation (Fig. 1). To analyze how this difference is encoded in the central complex of the beetle, we simultaneously presented the dung beetles with dorsal polarized UV light and an unpolarized green light spot while recording from early-stage compass cells (the same TL and CL1 cells as above). To imitate the natural polarization pattern of the sky, the electric field vectors of the zenithal polarizer were oriented perpendicular to the light spot’s azimuth, and the light intensity was adjusted to match either the conditions at low sun elevations or the conditions at low moon elevations. Because dung beetles seem to use a pure celestial compass system, without landmark information (8), this setup approximates the situation the orienting beetles would normally experience at low sun or moon elevations (Fig. 1A). When presenting both stimuli simultaneously to a TL neuron at brighter or lower light intensities corresponding to a low sun or moon elevation, the neuron of the diurnal beetle (Fig. S7A) ignored the polarization signals and responded exclusively to the green light spot. This neural response was also true for a TL neuron of the nocturnal beetle at brighter light intensities (Fig. 4A). However, when exposed to dimmer light intensities matching full moonlight, the TL neuron of the nocturnal species no longer responded to the artificial celestial body. Instead, the firing activity of the neuron was clearly modulated by the polarization channel (Fig. 4A).

To quantify which of the two stimuli dominates the neural responses under the two different light conditions, we combined a Gaussian fit (describing the response to the light spot) and a sine-square fit (describing the neural response to polarized light) in a single weighted fit function. This curve fit method enabled us to obtain a weighting factor between 0 and 1. A weighting factor between 0 and 0.5 indicates that the neuron responded with a 360° periodicity to the light stimulus (i.e., to the sun or moon azimuth), whereas a weighting factor between 0.5 and 1 indicates that the cells responded with a 180° periodicity (i.e., to the polarized light) (Fig. 4B). The results of this quantitative analysis matched exactly what we observed in the behavior of the beetles: When stimulated exclusively with polarized UV light, the spiking activity of central compass neurons was sinusoidally modulated, with a high average weighting factor of 0.79 ± 0.12 (mean ± SD) and 0.87 ± 0.06, no matter whether we tested diurnal or nocturnal beetles and irrespective of light intensity (Fig. 4C and Fig. S7B). Conversely, when we showed only a green light spot to the animal, the weighting factor was relatively low, again irrespective of species or light condition (Fig. 4C, between 0.21 ± 0.14 and 0.39 ± 0.08). When we stimulated with both cues simultaneously, the results depended upon the species. In the diurnal species, at both light intensities, the cells’ firing activity was modulated similar to the firing activity observed with the green light spot alone, suggesting that central complex neurons encode the sun or moon as the main celestial cue. In the nocturnal species, in contrast, this weighting was only the case at high light intensities, indicating that these neurons encode the sun’s position (Fig. 4C). However, when shown both cues at low light intensities (simulating a clear sky lit by a full moon), the firing activity of neurons in the nocturnal species was similar to the firing activity observed with polarized light stimulation alone (weighting factor: 0.75 ± 0.11; n = 5) showing that the neurons preferentially encode polarized skylight at this dimmer light level.

In summary, the neural encoding of celestial orientation cues in bright and dim light conditions closely matches the observed cue preference of these cues during the natural orientation behavior of our model species in the field (Fig. 5): Nocturnal dung beetles use the celestial polarization pattern as their main compass cue at night, whereas diurnal beetles use the sun as their main compass cue during the day.

Celestial Polarization Pattern: A Reliable Orientation Cue for Nocturnal Animals?

What then are the mechanisms that generate the switch to polarized light under dim light conditions in the nocturnal species? Studies in locusts and crickets have shown that the tuning of compass neurons to polarized light is intensity-independent above a particular threshold level of polarized light intensity (31, 32), whereas responses to unpolarized light strongly depend on light intensity (32). Consequently, when both stimuli are dimmed to lunar light intensities, only the response in the channel that is processed in an intensity-dependent manner should be diminished (i.e., the input from the light spot in these experiments). At lunar light intensities, the inputs should thus be modified in favor of the polarization channel for as long as the polarization analyzer of the eye is sensitive enough to detect a signal. In the nocturnal dung beetle, we found that the neurons responded reliably to bright as well as dim polarized light. The neuronal response in the diurnal species, on the other hand, was weak or absent at dim light intensities compared with the neuronal response at high light intensities (Fig. S6C). These neural responses might, at least partially, be a result of the size ratio between the dorsal rim area and main retina. The dorsal rim areas of crepuscular beetles are enormous (33), and our first preliminary observations suggest that this is also true for the nocturnal species tested here. In contrast, the dorsal rim areas of the diurnal species seem to be relatively small. The higher sensitivity to polarized light, and thus the weighting of different celestial cues in central complex neurons, might therefore be determined at an earlier stage in the brain. This difference in sensitivity to the polarization of light is also reflected in the orientation behavior of our two model species: Under dim light intensities, nocturnal navigators will adhere to the direction given by the polarization pattern rather than to the direction indicated by the moon’s position, whereas the diurnal species follows the direction derived from the moon (Fig. 5). This cue preference raises an important question regarding the advantages that a sky-wide polarization pattern offers over a bright point source (in the form of a moon) for nocturnal orientation. Because this difference in cue preference is a phenomenon observed only under dim light intensities, the answer is most likely a gain in sensitivity. One excellent strategy to gain visual sensitivity is to pool signals from many photoreceptors in one second-order neuron, a mechanism known as spatial summation (34, 35). The polarization pattern extends over the entire sky, and therefore allows the receiver to integrate signals over a wide visual field. At low light intensities, the polarization pattern should be a more reliable orientation cue than a moon, appearing as a dimly illuminated disk in the sky that may also be blocked from view by clouds. Moreover, even under the low light of a quarter or crescent moon, S. satyrus can consistently roll a straight-line path when the moon itself is blocked from view, almost certainly using the lunar polarization pattern for orientation (9). Similarly, crickets are able to detect polarized light even at light intensities equivalent to the light intensities of a moonless night sky (36). Under all these extremely dim conditions, spatial summation might be even more relevant for the orientation compass of nocturnal insects, as hypothesized by Labhart et al. (31); thus, the polarization pattern might offer the most reliable celestial reference for nocturnal navigation. Even though the moon, in any phase, is only available for about half of the month, it can still polarize the night sky when it is up to 15° below the horizon (37), extending the period when S. satyrus can reliably forage. Nevertheless, when the sun and moon are further below the horizon than 15°, and the night sky has no overall pattern of polarization, S. satyrus can fall back on the stars of the Milky Way to roll dung balls through the African bush (16), a cue that presumably ranks lowest in the cue preference of orientation signals.

In summary, our study clearly demonstrates how the visual ecology of an orienting animal is reflected in its orientation compass system. Here, the dynamic activity of neurons in the central complex provides the nocturnal orienting animal with a simple yet efficient tool to lock onto the most reliable signal available. This dynamic tuning generates a robust internal compass that can ensure precise orientation behavior with the cues available.

General.

Beetles were collected in South Africa. Details are provided in SI Materials and Methods.

Investigation of the Celestial Cue Preference.

To test which of the cues (sun/moon or polarization pattern) dominates the sky compass of dung beetles, we analyzed the rolling behavior with the celestial cues aligned or set in conflict (SI Materials and Methods).

Indoor Experiments with Polarized Light.

To analyze the wavelength range over which the beetles perceive polarized light, we presented zenithal polarized light in the UV or green range to the beetles in an indoor arena (SI Materials and Methods).

Orientation Under the Sun and a Green Light Spot.

To test whether beetles interpret a green light spot as the sun or moon, we compared the bearings taken with respect to the sun or moon with the bearings taken with respect to a green light spot indoors. Details are provided in SI Materials and Methods.

Physiology.

To analyze neural activity, we used a standard electrophysiology method to record responses of neurons intracellularly. Details are provided in SI Materials and Methods.

Visual Stimulations.

To test whether central complex neurons are sensitive to polarized UV light and/or to a green light spot, we designed a stimulus similar to the one described by Pfeiffer and Homberg (38). Details are provided in SI Materials and Methods.

Anatomy.

Immunohistochemistry, image acquisition, and 3D reconstruction procedures for neurons and neuropils have been described in detail by el Jundi et al. (39). Details are provided in SI Materials and Methods.

Data Analysis.

All experiments were analyzed with custom-written programs in MATLAB (MathWorks). Details are provided in SI Materials and Methods.

General.

All experiments were performed with adult dung beetles of the diurnal species S. (Kheper) lamarcki and the nocturnal species S. satyrus (Coleoptera; Scarabaeidae). Both species were collected in South Africa using baited pit-fall traps. They were kept outdoors in soil-filled plastic bins (30 × 22 × 22 cm) and were fed fresh cow dung daily. A net prevented the beetles from escaping but still allowed them to see the full sky at all hours of the day. Behavioral experiments were conducted in the beetles’ natural habitat on the game farm “Stonehenge” in North West province, South Africa (24.32°E, 26.39°S). Experiments were performed in January and November 2013 and 2014 at sun and moon elevations between 15° and 30°. Under these conditions, both the polarization pattern and the sun or moon are clearly visible to the animals.

For anatomical and electrophysiological studies, dung beetles of both species were transferred to Sweden, where they were housed in large boxes (50 × 36 × 27 cm) at the Department of Biology (Lund University). The day-active species was kept under a 12-h light/dark cycle, with lights on at 10:00 AM and at a room temperature of 26 °C. The nocturnal species was kept in a second animal room (12-h light/dark cycle), with an 11-h shifted light/dark cycle (lights off at 11:00 AM). These light/dark cycles allowed an easy analysis of the neural activity of the diurnal species during its subjective day and of the nocturnal species during its subjective night. Both species were fed with fresh cow dung collected from a farm close to Lund. Under these conditions, the beetles survived for up to 1 y.