Neural coding underlying the cue preference for celestial orientation

Basil el Jundia,1, Eric J. Warranta, Marcus J. Byrneb, Lana Khaldya, Emily Bairda, Jochen Smolkaa, and Marie Dackea,b

aDepartment of Biology, Lund University, 223 62 Lund, Sweden; and bSchool of , Plant, and Environmental Sciences, University of the Witwatersrand, Wits 2050, South Africa

Edited by John G. Hildebrand, University of Arizona, Tucson, AZ, and approved July 21, 2015 (received for review January 21, 2015)

Diurnal and nocturnal African dung use celestial cues, such different celestial cue preference than nocturnal species? If so, as the sun, the moon, and the polarization pattern, to roll dung how are these preferences represented neurally in the brain? balls along straight paths across the savanna. Although nocturnal In this study, we present a detailed picture of how the orientation beetles move in the same manner through the same environment systems of two closely related nocturnal and diurnal have as their diurnal relatives, they do so when light conditions are at been adapted to the ambient light conditions, combining behavioral least 1 million-fold dimmer. Here, we show, for the first time to experiments from the field with electrophysiological investigations of our knowledge, that the celestial cue preference differs between the underlying neural networks. Using behavioral experiments, we nocturnal and diurnal beetles in a manner that reflects their show that nocturnal dung beetles switch from a compass that uses a contrasting visual ecologies. We also demonstrate how these cue discrete celestial body (the sun) during the day to a celestial polari- zation compass for dim light orientation at night, whereas diurnal preferences are reflected in the activity of compass neurons in the beetles use a celestial body (the sun or moon) for orientation at all brain. At night, polarized skylight is the dominant orientation cue light levels. In a second step, we simulated these skylight cues (the for nocturnal beetles. However, if we coerce them to roll during sun or moon and the polarization pattern) while electrophysiologi- the day, they instead use a celestial body (the sun) as their primary cally recording responses from neurons in the dung ’scentral orientation cue. Diurnal beetles, however, persist in using a complex, a brain area that has been suggested to house the internal celestial body for their compass, day or night. Compass neurons compass for celestial orientation (13, 14). These neural data precisely in the central complex of diurnal beetles are tuned only to the matched the cue preferences observed in behavioral field trials and sun, whereas the same neurons in the nocturnal species switch show how an animal’s visual ecology influences the neural activity of exclusively to polarized light at lunar light intensities. Thus, these itsskycompassneurons.Ourresults also reveal, for the first time to neurons encode the preferences for particular celestial cues and alter our knowledge, how a weighting of celestial orientation cues could be their weighting according to ambient light conditions. This flexible neurally encoded in an animal brain. encoding of celestial cue preferences relative to the prevailing visual scenery provides a simple, yet effective, mechanism for enabling Results and Discussion visual orientation at any light intensity. Nocturnal Dung Beetles Use the Polarization Pattern as a Primary Orientation Cue at Night. Dung beetles use a large repertoire of navigation | | vision | central complex | dim light celestial cues for orientation (5, 7, 15–17), but their two main compass cues are (i) the sun or moon and (ii) the celestial po- he blue sky is a rich source of visual cues that are used by larization pattern (5). Here, we first sought to test if one compass many animals during orientation or navigation (1, 2). Besides cue is dominant over the other in the orientation systems of two T closely related species that are active at different the sun, celestial phenomena, such as the skylight intensity gra- times of the day: Scarabaeus lamarcki (diurnal) and Scarabaeus dient or the more complex polarization pattern, can serve as ref- satyrus (nocturnal). The robust straight-line orientation behavior of erences for spatial orientation (3–5). Polarized skylight is generated by scattered sunlight in the atmosphere, and to a terrestrial ob- Significance server, the resulting alignment of the electric field vectors extends across the entire sky, forming concentric circles around the position of the sun (Fig. 1A). A similar distribution of brightness and po- Many animals use the sun or moon and the polarization pat- tern for navigation. We combined behavioral experiments with larization pattern is also created around the moon (6). Although physiological measurements of brain activity to reveal which this nocturnal pattern is 1 million-fold dimmer than the daylight celestial cue dominates the orientation compass of diurnal and pattern (6), some animals, such as South African ball-rolling dung nocturnal dung beetles. The preference found behaviorally beetles, can use this lunar polarization pattern for orientation (7). precisely matches the preference encoded neurally and shows To avoid competition for food at the dung pile, these beetles detach how the brain dynamically controls the cue preference for a piece of dung, shape it into a ball, and roll it away along a straight- orientation at different levels: The sun or moon always domi- line path. For this type of straight-line orientation, nocturnal beetles nates the orientation behavior and neural tuning of diurnal seem to rely exclusively on celestial cues (8), such as the moon or beetles, whereas in nocturnal beetles, celestial bodies domi- polarized light. nate tuning only in bright light, with a switch to polarized light As with all nocturnal animals, night-active beetles have to at night. This flexible neural tuning in the nocturnal species overcome a major challenge: They need to maintain high ori- provides a simple mechanism that allows it to use the most entation precision even under extremely dim light conditions. reliable available orientation cue. Indeed, recent experiments have shown that nocturnal dung beetles orient at night with the same precision as their diurnal Author contributions: B.e.J., E.J.W., M.J.B., E.B., J.S., and M.D. designed research; B.e.J., relatives during the day (9), an ability partly due to the fact that M.J.B., L.K., E.B., and M.D. performed research; B.e.J. and J.S. analyzed data; and B.e.J. their eyes are considerably more sensitive than the eyes of spe- wrote the paper. cies that are active at brighter light levels (10–12). Nonetheless, The authors declare no conflict of interest. for each species, orientation precision relies on being tuned to This article is a PNAS Direct Submission. the most reliable celestial compass cue that is available during 1To whom correspondence should be addressed. Email: [email protected]. ’ PHYSIOLOGY the animal s normal activity window. How salient are these cues This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. for nocturnal and diurnal species? Do diurnal species have a 1073/pnas.1501272112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1501272112 PNAS | September 8, 2015 | vol. 112 | no. 36 | 11395–11400 Downloaded by guest on October 1, 2021 still be visible (and unaltered) across the remainder of the visible A 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 sun/moon 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 B 0° 0° significantly toward the opposite sky hemisphere), both during the Change of direction day (P < 0.001 by V test,withanexpectedmeanof180°)andat = -90° D 90° -90° N 90° night (P 0.002 by V test,withanexpectedmeanof180°)(Fig.1B and C, Left and Figs. S1A and S2 A and C). This result matches our control 180° 180° previous findings (5, 17, 18) and suggests that diurnal dung beetles test 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 C 0° also true for the nocturnal species when orienting during the day Change of direction 0° [Fig. 1B, Right (P < 0.001 by V test, with an expected mean of 180°) D and Figs. S1A and S2B]. However, when these nocturnal foragers -90° 90° -90° N 90° rolled their balls in the light of the full moon, they displayed a control different strategy: The beetles did not change their rolling direction 180° 180° = test (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 Fig. 1. Celestial cue preference in dung beetles under a natural sky. (A)Sche- was reflected back at them from the opposite direction [Fig. 1C, matic illustration of the polarization pattern around a celestial body (sun or Right (P < 0.001 by V test, with an expected mean of 0°) and Figs. moon). Change of direction in diurnal (D, Left) and nocturnal (N, Right) S1A and S2D]. In contrast to the diurnal species, the nocturnal beetles rolling under a sun-lit (B) or moon-lit (C) sky. The change of direction species thus primarily followed the direction given by the skylight was calculated as the angular difference between two consecutive rolls, ei- polarization pattern under dim light conditions. To confirm this ther without manipulation (control, ●) or when the sun or moon was observation, we also tested the nocturnal species rolling under a reflected to the opposite sky hemisphere between the two rolls (test, ○). The polarizing filter with a clear view of the full moon (elevation <30°) mean directions (μ) are indicated by black (control) or red (test) lines, and through the polarizer. The polarizer was placed on top of a error bars indicate circular SDs. (B) Without manipulation, both species kept screened arena, with the filter’s E-vectors either aligned (Fig. S3A) the direction [P < 0.001 by V test; μdiurnal (±SD) = 2.6° ± 17.98°, n = 20; μ = − ± = or in conflict (Fig. S3B) with the lunar polarized skylight. In contrast nocturnal 8.7° 38.34°, n 20). When the sun was reflected to the op- to the diurnal species, which orients with respect to the sun even if posite sky hemisphere (and the real sun was shaded), both species responded the polarizer is turned by 90° (5), the nocturnal species significantly to this change (P < 0.001 by V-test; μdiurnal = 178.9° ± 54.6°, n = 20; μnocturnal = 163.8° ± 46.58°, n = 20). (C) Under the moon in the control experiments, changed its bearing in response to a change in the polarized light direction (Fig. S3). This experiment also shows that polarized light both species showed a constant rolling direction (P < 0.001 by V test; μdiurnal = 3.1° ± 35.39°, n = 20; μnocturnal = −3.3° ± 37.87°, n = 20). When the moon was is ranked higher than the disk of the moon in the internal compass reflected to the opposite sky hemisphere, the diurnal species followed the of nocturnal dung beetles. Overall, we show that despite the taxo- position change of the moon (P = 0.002 by V test; μ = 179.5° ± 72.37°, n = 20), nomic proximity of both species, they rely on different celestial cues whereas the nocturnal species continued rolling in the original rolling direction for their dominant directional reference under dim light conditions. (P < 0.001 by V test; μ = −13.4° ± 74.27°, n = 20). 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 these beetles can be induced independent of the time of day, observed switch to a polarization compass in the nocturnal species. allowing us to examine the beetles’ choice of celestial cues even at times when they are not usually active (i.e., the nocturnal species Central Complex Cells Are Sensitive to Polarized UV Light. Next, we during the day, the diurnal species at night). First, we measured the sought to understand how the polarized light pattern and the po- change in the beetles’ directions between two consecutive rolls when sition of the sun or moon are processed neurally in the beetle’s they rolled their balls out of an arena (1-m diameter) on a sunlit day brain. To allow an accurate investigation of the neural substrate for or under a clear, moonlit sky (Fig. 1 B and C; control, ●). We tested polarization-dependent orientation, we first had to define at which the beetles’ orientation behavior at low sun elevations or (on full- wavelength of light the beetles perceive the skylight polarization moon nights) moon elevations (<30°). Under these conditions, a pattern. Dacke et al. (15) suggested that dung beetles detect po- celestial body (sun or moon) and the celestial polarization pattern larized light either in the green or UV wavelength. Therefore, we created by it are both clearly visible to the animals (Fig. 1A). In all investigated the orientation performance of both of our model cases, the change of direction between the two repetitions was species in an indoor arena lit by polarized light from above, with a clustered around 0° (P < 0.001 by V test,withanexpectedmeanof peak either in the UV (365 nm) or green (530 nm) wavelength (Fig. 0°); that is, the beetles did not change their bearing between con- 2 A and B and Fig. S4). Each beetle was made to roll a ball twice secutive rolls (Fig. 1 and Figs. S1A and S2 A–D). This observation from the center to the perimeter of a circular indoor arena. Be- confirmed that both species have a consistent orientation behavior tween each roll, the polarizer either remained in place (control) or even at times when they are not naturally active. was turned by 90° (test). Under polarized UV light, both species To investigate the relative importance of, or preference for, changed their heading by 90° [Fig. 2B (inbothspecies,P < 0.001 by the two main celestial cues in the beetles’ compass system (sun or V test,withanexpectedmeanof90°)andFigs. S1B and S2 E and moon vs. polarized light), we also examined how beetles reacted F], whereas under polarized green light, the beetles were dis- when the two cues were set in conflict. Again, beetles were made oriented (Figs. S1B and S4). Thus, as in many other (19–22), to roll out from the center of the arena on two consecutive oc- both of our dung beetle species detect the skylight polarization casions: once in the light of the sun or the moon and once with pattern in the UV range. the sun or full moon (celestial body elevations <30°) “displaced” To analyze the tuning of the polarization-sensitive neurons, we to the opposite sky hemisphere using a mirror (at the start of presented the beetles with zenithal polarized UV light that had either the first roll or the second roll), while simultaneously similar light intensities as in the behavioral experiments. Before hiding the real sun or moon from the beetle’s view (Fig. 1 B and starting to roll, dung beetles always climb on top of their balls and C; test, ○). If the beetle relies on polarized skylight as its main perform “orientation dances” in which they rotate around their cue, it should adhere to its original rolling direction, which would own body axis (23). To simulate this vertical rotation underneath

11396 | www.pnas.org/cgi/doi/10.1073/pnas.1501272112 el Jundi et al. Downloaded by guest on October 1, 2021 AB C

D

Fig. 2. Analysis of the polarization-dependent orientation systems in dung beetles. (A) Schematic drawing of the experimental design simulating the po- larization pattern. The roll bearings of both species were analyzed with respect to zenithal polarized light in an indoor arena. (B) Response of diurnal (Left) and nocturnal (Right) dung beetles to polarized UV light. The angular difference between two rolls was recorded when the polarizer remained in place (control, black circles) or after a 90° turn of the polarizer (test, magenta circles). Both species oriented in response to the polarizer; that is, their changes of direction were clustered along the 0 to 180° axis in the control conditions (P < 0.001 by V test, n = 20 for each species) and oriented along the 90 to −90° axis when the polarizer was turned (P < 0.001 by V test, n = 20 for each species). The mean directions are indicated by gray (control) or magenta (test) lines, and error bars indicate circular SD. (C) Neural response of a TL neuron to a 360° revolution of a zenithal polarizer. (Middle) Firing activity of the neuron. (Upper) Sliding average of the tuning. (Lower) Bar indicates the rotation of the polarizer, and the disk shows a schematic drawing of the polarizer with E-vector orientation (double-headed arrows) at the initial point of rotation. (D) Neural tuning of four central complex cell types. The circular diagrams show the neurons’ firing activities plotted against stimulus orientation (in 10° bins). These plots are shown from the beetle’s point of view, with 0° in front of the

animal. All neurons responded to the rotating polarizer [Rayleigh test: TL, P < 0.001 (nrotations = 2); CL1, P < 0.001 (nrotations = 10); TB1, P < 0.01 (nrotations = 10); CPU1, P < 0.01 (nrotations = 4)]. Error bars show SD. Black circles show background spiking activities. Red lines indicate the mean direction of the neuron.

the natural polarization pattern, we rotated the polarizer by 360° (or moon), as has recently been shown for locusts and monarch while recording from compass neurons of the central complex. butterflies (26, 27). The dung beetle’s central complex consists of four brain areas, which have been described in detail in other insects (24): the Central Complex Neurons also Encode the Sun/Moon Azimuth. During upper and lower divisions of the central body (termed the fan- our experiments, we also analyzed how the very same neurons that shaped and ellipsoid body in flies), the protocerebral bridge, and responded to polarized light were modulated in response to a the paired noduli (Fig. S5 A–C). moving unpolarized green light stimulus. In honey bees, a green Using single-cell tracer injections, we were able to identify (and light spot is interpreted as the sun’sdirection(28–30). To test record from) all four types of central complex neurons known to whether the sun or moon can be replaced by an artificial green light process polarization signals in other orienting insects (14) (Fig. spot also in the compass system of the beetles, we let the beetles roll S5D). According to a proposed processing network in the locust’s first under the clear sky and then again immediately afterward in an central complex (25), these cells can be categorized into early and indoor arena, lit only by a green light spot (or vice versa, using the late processing-stage neurons: Input tangential (TL) neurons and light spot first followed by the celestial cue) (Fig. 3 A and B). In- columnar (CL1) neurons represent the early processing stage, terestingly, even when the beetles had a green light spot as their sole whereas tangential (TB1) cells and columnar (CPU1) neurons orientation cue, their orientation was equally precise (nocturnal represent the late processing stage (Fig. S5D). In total, we recorded species) or only slightly less precise (diurnal species) than their from 21 central complex neurons [TL: n = 8 (three diurnal, five performance outdoors when many celestial cues were available (Fig. nocturnal), CL1: n = 9 (four diurnal, five nocturnal); TB1: n = 3 S1C). We found that irrespective of whether the beetles (n = 20 (one diurnal, two nocturnal); CPU1: n = 1 (nocturnal)] while diurnal beetles and n = 20 nocturnal beetles) first rolled under the rotating the polarizer by 360° above each beetle at least twice (one green light spot (elevation about 25°) or under the sun or moon, the clockwise rotation and one counterclockwise rotation) (Dataset S1). beetles’ heading was maintained in both situations (i.e., the change Neurons of the same type showed similar responses, independent of direction was clustered around 0°) (Fig. 3C;inbothspecies,P < of the species’ identity. When stimulating early processing-stage 0.001 by V test, with an expected mean of 0°; Fig. S2 G and H). Both cells (TL and CL1) with a rotating polarized UV light, the neurons dung beetle species thus clearly orient in a similar way to a green were maximally excited at a particular orientation of the polarizer light spot as they do to a celestial disk. and were maximally inhibited at the perpendicular orientation (Fig. Next, we simulated beetles rotating under the sun or the moon 2 C and D), demonstrating their angular sensitivity to polarized UV (but in the absence of a celestial polarization pattern) and ana- light. In contrast, late processing-stage cells (TB1 and CPU1) typ- lyzed the response of 20 neurons to a moving green light spot ically showed a firing activity that was similarly, but less strongly, that moved on a circular path around the beetle’s head. In total, modulated during polarizer rotation (Fig. 2D and Fig. S6 A and B). 19 (TL: n = 7, CL1: n = 9, TB1: n = 3) of the polarization- This neural tuning is well in line with observations from the locust’s sensitive central complex neurons recorded above showed a central complex and can most likely be explained by a multimodal significant response also to the moving green light spot (Fig. 3 D function of the late processing cells in the central complex network and E; P < 0.05 by Rayleigh test) and were clearly modulated by (14, 25). In summary, the dung beetle’scentralcomplexactsasakey the stimulus position (Fig. S6 A and B). Early processing-stage cells processing center for polarized light analysis. However, to function (TL and CL1) were excited by the green light spot when it passed a as a robust internal compass for straight-line orientation, it would certain position in the visual field. TL neurons showed an excitation PHYSIOLOGY also have to identify and integrate the azimuthal position of the sun with a narrow receptive field. In contrast, CL1 neurons showed an

el Jundi et al. PNAS | September 8, 2015 | vol. 112 | no. 36 | 11397 Downloaded by guest on October 1, 2021 ABC

DE

Fig. 3. Analysis of the azimuth-dependent orientation systems in dung beetles. (A) Schematic illustration of the experimental setup. The roll bearings of beetles were analyzed with respect to a green light spot in an indoor arena and compared with the rolling directions with respect to the sun (diurnal species) or moon (nocturnal species) outdoors. The elevation of the light spot and the celestial body was similar (<30°). (B) Beetles rolled their balls three times under a celestial body (yellow dots) and three times with a light spot (green dots) as their only orientation cue. The angular difference between the mean direction relative to the celestial body (yellow line) and the light spot (green line) was measured as the change of direction (black sector). (C)Indiurnal(Left, n = 20) and nocturnal (Right, n = 20) beetles, the change

ofdirectionwasclusteredaround0°[P < 0.001 by V test; μdiurnal (±SD) = −24.5° ± 46.25°; μnocturnal = −4.6° ± 37.23°]. Mean direction is shown as a green line, and error bars show SD. (D) Neural activity of a CL1 cell to a green light spot moved on a circular path around the beetle’s head (elevation of 30°). (Middle) Firing activity of the neuron. (Upper) Sliding average of the tuning. (Lower) Solid line shows the position of the light spot with respect to the beetle’s head, and the schematic drawing shows the position of the light spot at the initial position relative to the animal. (E) Firing activity of three central complex cells (recorded in both species) to a light spot. The circular plots show the neuron’s firing activity plotted against stimulus orientation (in 10° bins). All plots are shown from the beetle’s point of view, with 0° in front

of the animal. The cells respond significantly to the green light spot [Rayleigh tests: TL; P < 0.01 (nrotations = 2); CL1, P < 0.001 (nrotations = 4); TB1, P < 0.001 (nrotations = 2)]. Error bars show SD. Black circles show background firing activities. Red lines indicate the mean direction of the neuron.

additional strong inhibition in the opposite visual field (Fig. 3E). light intensities (Fig. 4A). However, when exposed to dimmer light Two CL1 neurons showed only inhibition as a response to the intensities matching full moonlight, the TL neuron of the noc- green light spot. In contrast, the response characteristics of late turnal species no longer responded to the artificial celestial body. processing-stage (TB1) cells were diverse but always featured an Instead, the firing activity of the neuron was clearly modulated by increased average neural activity at a certain azimuth of the light the polarization channel (Fig. 4A). spot (Fig. 3E). In summary, our data show that compass neurons To quantify which of the two stimuli dominates the neural re- in the beetle’s central complex encode the pattern of polarized sponses under the two different light conditions, we combined a skylight and are suited to encode the sun’s or moon’s azimuth Gaussian fit (describing the response to the light spot) and a sine- (especially cells at an early processing stage). In combination, square fit (describing the neural response to polarized light) in a these two cues can be used to generate a robust internal sky single weighted fit function. This curve fit method enabled us to compass signal for straight-line orientation. Next, to test the obtain a weighting factor between 0 and 1. A weighting factor be- relative representations of both signals at the neural level, we set tween0and0.5indicatesthatthe neuron responded with a 360° out to analyze the neural activity of central complex cells when periodicity to the light stimulus (i.e., to the sun or moon azimuth), the animal was exposed to both stimuli simultaneously. 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. Tuning of Central Complex Neurons Matches the Beetles’ Behavioral 4B). The results of this quantitative analysis matched exactly what we Cue Preference. Behaviorally, we found that diurnal dung beetles observed in the behavior of the beetles: When stimulated exclusively primarily use a celestial body for orientation, both during the day with polarized UV light, the spiking activity of central compass and at night. Nocturnal dung beetles also use this cue under daylight neurons was sinusoidally modulated, with a high average weighting intensities but switch to a polarization compass for nocturnal ori- factor of 0.79 ± 0.12 (mean ± SD) and 0.87 ± 0.06, no matter entation (Fig. 1). To analyze how this difference is encoded in the whether we tested diurnal or nocturnal beetles and irrespective of central complex of the beetle, we simultaneously presented the light intensity (Fig. 4C and Fig. S7B). Conversely, when we showed dung beetles with dorsal polarized UV light and an unpolarized only a green light spot to the animal, the weighting factor was rela- green light spot while recording from early-stage compass cells (the tively low, again irrespective of species or light condition (Fig. 4C, same TL and CL1 cells as above). To imitate the natural polari- between 0.21 ± 0.14 and 0.39 ± 0.08).Whenwestimulatedwithboth zation pattern of the sky, the electric field vectors of the zenithal cues simultaneously, the results depended upon the species. In the polarizer were oriented perpendicular to the light spot’sazimuth, diurnal species, at both light intensities, the cells’ firing activity was and the light intensity was adjusted to match either the conditions at modulated similar to the firing activity observed with the green light low sun elevations or the conditions at low moon elevations. Be- spot alone, suggesting that central complex neurons encode the sun cause dung beetles seem to use a pure celestial compass system, or moon as the main celestial cue. In the nocturnal species, in con- without landmark information (8), this setup approximates the trast, this weighting was only the case at high light intensities, in- situation the orienting beetles would normally experience at low dicating that these neurons encode the sun’s position (Fig. 4C). sun or moon elevations (Fig. 1A). When presenting both stimuli However, when shown both cues at low light intensities (simulating a simultaneously to a TL neuron at brighter or lower light intensities clear sky lit by a full moon), the firing activity of neurons in the corresponding to a low sun or moon elevation, the neuron of the nocturnal species was similar to the firing activity observed with diurnal beetle (Fig. S7A) ignored the polarization signals and polarized light stimulation alone (weighting factor: 0.75 ± 0.11; n = 5) responded exclusively to the green light spot. This neural response showing that the neurons preferentially encode polarized skylight at was also true for a TL neuron of the nocturnal beetle at brighter this dimmer light level.

11398 | www.pnas.org/cgi/doi/10.1073/pnas.1501272112 el Jundi et al. Downloaded by guest on October 1, 2021 A 30 bright N B 25 C N D

)s/pmi(ytivitcanaem weight: 0.34 1.0 bright dim bright dim 20 )s/pmi

15 L

O 0.8

10 P

th

(ytivitc 5 0.6

g

0 iew 30 25 weight: 0.87 to 0.4 dim ps

an

t

20 15 hgiL 0.2

a 10 em 5 0.0

sul su 0 10 9 7 5 5 5 7 7 6 6 6 6

lumits

u

0° 360° -90° 270° 0° 360° 0° 360° mits -90° 270° -90° 270°

Fig. 4. Decoding the celestial cue preference in the dung beetle’s central complex. (A, Upper) Sliding average of the response of a TL neuron to a rotating polarizer (Left, magenta), a light spot (Middle, green), and when both stimuli were shown simultaneously (Right, black) at light intensities that simulated conditions at low sun elevations. Gray curves show individual rotations. (A, Lower) Sliding average of the response of the same neuron, with light intensities that simulated conditions of a full-moon–lit night. Solid lines show stimulus timing, and disks and circles indicate stimulus type (Figs. 2 and 3). (B) Mean spiking activity (bin size of 10°) of the same neuron when both stimuli were shown simultaneously at high light intensity (Upper) and low light intensity (Lower) (same responses as in A, Right, black). The solid curve shows the combined Gaussian fit and sine-square fit. The weighting factor indicates how well the neural tuning was described by a Gaussian fit (between 0 and 0.5, response to the moving light spot) or a sine-square fit (between 0.5 and 1, response to rotating polarizer) function. Error bars show SD. (C) Weights of the neural response to different light stimuli. (Left) Neural responses of central complex neurons (TL and CL1) of the nocturnal species to polarized (POL) light (Left;TL,n = 5; CL1, n = 5), a light spot (Middle;TL,n = 4; CL1, n = 5), or both stimuli presented simultaneously (Right, red; TL, n = 4; CL1, n = 3). A white background denotes high light intensities, and a gray background denotes full-moon light conditions (TL, n = 3; CL1, n = 2). Numbers represent cells analyzed. Error bars show SD. (Right) Same, but for cells analyzed in the diurnal species (TL, n = 3; CL1, n = 4).

In summary, the neural encoding of celestial orientation cues in strategy to gain visual sensitivity is to pool signals from many pho- bright and dim light conditions closely matches the observed cue toreceptors in one second-order neuron, a mechanism known as preference of these cues during the natural orientation behavior of spatial summation (34, 35). The polarization pattern extends over our model species in the field (Fig. 5): Nocturnal dung beetles use the entire sky, and therefore allows the receiver to integrate signals the celestial polarization pattern as their main compass cue at night, over a wide visual field. At low light intensities, the polarization whereas diurnal beetles use the sun as their main compass cue pattern should be a more reliable orientation cue than a moon, during the day. 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 Celestial Polarization Pattern: A Reliable Orientation Cue for a quarter or crescent moon, S. satyrus can consistently roll a straight- Nocturnal Animals? What then are the mechanisms that generate line path when the moon itself is blocked from view, almost cer- the switch to polarized light under dim light conditions in the tainly using the lunar polarization pattern for orientation (9). Sim- nocturnal species? Studies in locusts and crickets have shown that ilarly, crickets are able to detect polarized light even at light the tuning of compass neurons to polarized light is intensity- intensities equivalent to the light intensities of a moonless night sky independent above a particular threshold level of polarized light (36). Under all these extremely dim conditions, spatial summation intensity (31, 32), whereas responses to unpolarized light strongly might be even more relevant for the orientation compass of noc- depend on light intensity (32). Consequently, when both stimuli are turnal insects, as hypothesized by Labhart et al. (31); thus, the po- dimmed to lunar light intensities, only the response in the channel larization pattern might offer the most reliable celestial reference that is processed in an intensity-dependent manner should be di- for nocturnal navigation. Even though the moon, in any phase, is minished (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 in- tensities compared with the neuronal response at high light in- tensities (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 de- termined at an earlier stage in the brain. This difference in sensi- Fig. 5. Summary of results. The illustration of the ball-rolling dung beetle tivity to the polarization of light is also reflected in the orientation represents the behavioral experiments. The schematic drawing of the cell with behavior of our two model species: Under dim light intensities, the recording trace represents the physiological experiments. Diurnal species nocturnal navigators will adhere to the direction given by the po- (Left) and nocturnal species (Right) are shown. Both were analyzed at low sun larization pattern rather than to the direction indicated by the elevation (behavior) or with light conditions simulating low sun elevation moon’s position, whereas the diurnal species follows the direction (physiology) (Upper) and at low moon elevations (behavior) or with light simulating low moon elevation (physiology) (Lower). The diurnal species uses derived from the moon (Fig. 5). This cue preference raises an im- the celestial body as its primary orientation cue during both day and night, but portant question regarding the advantages that a sky-wide polari- the nocturnal species switches to the polarization pattern as its primary cue when zation pattern offers over a bright point source (in the form of a rolling balls at night. The behavioral demonstration of the compass cue prefer- moon) for nocturnal orientation. Because this difference in cue ence accurately matches the neural encoding of the celestial cue weighting PHYSIOLOGY preference is a phenomenon observed only under dim light in- in the compass neurons of the brain. In each panel, pale gray cues indicate tensities, the answer is most likely a gain in sensitivity. One excellent that this cue is not used as the main reference in this condition.

el Jundi et al. PNAS | September 8, 2015 | vol. 112 | no. 36 | 11399 Downloaded by guest on October 1, 2021 only available for about half of the month, it can still polarize the Orientation Under the Sun and a Green Light Spot. To test whether beetles night sky when it is up to 15° below the horizon (37), extending interpret a green light spot as the sun or moon, we compared the bearings the period when S. satyrus can reliably forage. Nevertheless, when taken with respect to the sun or moon with the bearings taken with the sun and moon are further below the horizon than 15°, and the respect to a green light spot indoors. Details are provided in SI Materials night sky has no overall pattern of polarization, S. satyrus can fall and Methods. 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 Physiology. To analyze neural activity, we used a standard electrophysiology preference of orientation signals. method to record responses of neurons intracellularly. Details are provided in In summary, our study clearly demonstrates how the visual SI Materials and Methods. ecology of an orienting animal is reflected in its orientation compass system. Here, the dynamic activity of neurons in the central complex Visual Stimulations. To test whether central complex neurons are sensitive to provides the nocturnal orienting animal with a simple yet efficient polarized UV light and/or to a green light spot, we designed a stimulus similar tool to lock onto the most reliable signal available. This dynamic to the one described by Pfeiffer and Homberg (38). Details are provided in SI tuning generates a robust internal compass that can ensure precise Materials and Methods. orientation behavior with the cues available. Anatomy. Immunohistochemistry, image acquisition, and 3D reconstruction Materials and Methods procedures for neurons and neuropils have been described in detail by el General. Beetles were collected in South Africa. Details are provided in SI Jundi et al. (39). Details are provided in SI Materials and Methods. Materials and Methods. Data Analysis. All experiments were analyzed with custom-written programs Investigation of the Celestial Cue Preference. To test which of the cues (sun/ in MATLAB (MathWorks). Details are provided in SI Materials and Methods. moon or polarization pattern) dominates the sky compass of dung beetles, we analyzed the rolling behavior with the celestial cues aligned or set in ACKNOWLEDGMENTS. We thank Drs. James Foster, Stanley Heinze, Octavian conflict (SI Materials and Methods). Knoll, Keram Pfeiffer, and Christine Scholtyssek for their helpful discussions and comments. We are grateful to Ted and Winnie Harvey for their invaluable help in the field. We also thank Drs. Erich Buchner and Christian Wegener for Indoor Experiments with Polarized Light. To analyze the wavelength range providing the antisynapsin antibody. Funding for this project was provided by over which the beetles perceive polarized light, we presented zenithal po- Vetenskapsrådet, the Wallenberg Foundation, the Wenner-Gren-Foundation, larized light in the UV or green range to the beetles in an indoor arena (SI the Royal Physiographic Society in Lund, the Lars-Hierta Memorial Foundation, Materials and Methods). and the South African National Research Foundation.

1. Wehner R (1984) Astronavigation in insects. Annu Rev Entomol 29:277–298. 22. Wernet MF, et al. (2012) Genetic dissection reveals two separate retinal substrates for 2. Wiltschko W, Wiltschko R (1991) Magnetic orientation and celestial cues in migratory polarization vision in Drosophila. Curr Biol 22(1):12–20. orientation. Orientation in Birds, ed Berthold P (Birkhäuser, Basel), pp 16–37. 23. Baird E, Byrne MJ, Smolka J, Warrant EJ, Dacke M (2012) The dung beetle dance: An 3. Frisch KV (1949) Die Polarisation des Himmelslichtes als orientierender Faktor bei den orientation behaviour? PLoS One 7(1):e30211. Tänzen der Bienen. Experientia 5(4):142–148. German. 24. Ito K, et al.; Insect Brain Name Working Group (2014) A systematic nomenclature for 4. Horváth G, Varjú D (2004) Polarized Light in Animal Vision: Polarization Patterns in the insect brain. Neuron 81(4):755–765. Nature (Springer, Heidelberg). 25. Heinze S, Gotthardt S, Homberg U (2009) Transformation of polarized light in- 5. el Jundi B, Smolka J, Baird E, Byrne MJ, Dacke M (2014) Diurnal dung beetles use the formation in the central complex of the locust. J Neurosci 29(38):11783–11793. intensity gradient and the polarization pattern of the sky for orientation. J Exp Biol 26. Heinze S, Reppert SM (2011) Sun compass integration of skylight cues in migratory 217(Pt 13):2422–2429. monarch butterflies. Neuron 69(2):345–358. 6. Gál J, Horváth G, Barta A, Wehner R (2001) Polarization of the moonlit clear night sky 27. el Jundi B, Pfeiffer K, Heinze S, Homberg U (2014) Integration of polarization and measured by full-sky imaging polarimetry at full Moon: Comparison of the polari- chromatic cues in the insect sky compass. J Comp Physiol A Neuroethol Sens Neural zation of the moonlit and sunlit skies. J Geophys Res 106(D19):22647–22653. Behav Physiol 200(6):575–589. 7. Dacke M, Nilsson DE, Scholtz CH, Byrne M, Warrant EJ (2003) Animal behaviour: Insect 28. Brines ML, Gould JL (1979) Bees have rules. Science 206(4418):571–573. orientation to polarized moonlight. Nature 424(6944):33. 29. Edrich W, Neumeyer C, von Heiversen O (1979) “Anti-sun orientation” of bees with 8. Dacke M, Byrne M, Smolka J, Warrant E, Baird E (2013) Dung beetles ignore land- marks for straight-line orientation. J Comp Physiol A Neuroethol Sens Neural Behav regard to a field of ultraviolet light. J Comp Physiol A Neuroethol Sens Neural Behav – Physiol 199(1):17–23. Physiol 134(2):151 157. 9. Dacke M, Byrne MJ, Baird E, Scholtz CH, Warrant EJ (2011) How dim is dim? Precision 30. Rossel S, Wehner R (1984) Celestial orientation in bees: the use of spectral cues. of the celestial compass in moonlight and sunlight. Philos Trans R Soc Lond B Biol Sci J Comp Physiol A Neuroethol Sens Neural Behav Physiol 155(5):605–613. 366(1565):697–702. 31. Labhart T, Petzold J, Helbling H (2001) Spatial integration in polarization-sensitive 10. McIntyre P, Caveney S (1998) Superposition optics and the time of flight in onitine interneurones of crickets: A survey of evidence, mechanisms and benefits. J Exp Biol dung beetles. J Comp Physiol A Neuroethol Sens Neural Behav Physiol 183:45–60. 204(Pt 14):2423–2430. 11. Greiner B, et al. (2007) Eye structure correlates with distinct foraging-bout timing in 32. Kinoshita M, Pfeiffer K, Homberg U (2007) Spectral properties of identified polarized- primitive ants. Curr Biol 17(20):R879–R880. light sensitive interneurons in the brain of the desert locust Schistocerca gregaria. 12. Warrant EJ (2008) Seeing in the dark: Vision and visual behaviour in nocturnal bees J Exp Biol 210(Pt 8):1350–1361. and wasps. J Exp Biol 211(Pt 11):1737–1746. 33. Warrant E (2004) Vision in the dimmest habitats on earth. J Comp Physiol A Neuroethol 13. Heinze S, Homberg U (2007) Maplike representation of celestial E-vector orientations Sens Neural Behav Physiol 190(10):765–789. in the brain of an insect. Science 315(5814):995–997. 34. Dacke M, Nordström P, Scholtz CH (2003) Twilight orientation to polarised light in the 14. Homberg U, Heinze S, Pfeiffer K, Kinoshita M, el Jundi B (2011) Central neural coding crepuscular dung beetle Scarabaeus zambesianus. J Exp Biol 206(Pt 9):1535–1543. – of sky polarization in insects. Philos Trans R Soc Lond B Biol Sci 366(1565):680 687. 35. Warrant EJ (1999) Seeing better at night: Life style, eye design and the optimum 15. Dacke M, Byrne MJ, Scholtz CH, Warrant EJ (2004) Lunar orientation in a beetle. Proc strategy of spatial and temporal summation. Vision Res 39(9):1611–1630. – Biol Sci 271(1537):361 365. 36. Herzmann D, Labhart T (1989) Spectral sensitivity and absolute threshold of polari- 16. Dacke M, Baird E, Byrne M, Scholtz CH, Warrant EJ (2013) Dung beetles use the Milky zation vision in crickets: A behavioral study. J Comp Physiol A Neuroethol Sens Neural Way for orientation. Curr Biol 23(4):298–300. Behav Physiol 165(3):315–319. 17. Dacke M, el Jundi B, Smolka J, Byrne M, Baird E (2014) The role of the sun in the 37. Cronin TW, Warrant EJ, Greiner B (2006) Celestial polarization patterns during twi- celestial compass of dung beetles. Philos Trans R Soc Lond B Biol Sci 369(1636): light. Appl Opt 45(22):5582–5589. 20130036. 38. Pfeiffer K, Homberg U (2007) Coding of azimuthal directions via time-compensated 18. Byrne M, Dacke M, Nordström P, Scholtz C, Warrant E (2003) Visual cues used by ball- – rolling dung beetles for orientation. J Comp Physiol A Neuroethol Sens Neural Behav combination of celestial compass cues. Curr Biol 17(11):960 965. Physiol 189(6):411–418. 39. el Jundi B, et al. (2009) The locust standard brain: A 3D standard of the central 19. Labhart T, Meyer EP (1999) Detectors for polarized skylight in insects: A survey of complex as a platform for neural network analysis. Front Syst Neurosci 3:21. ommatidial specializations in the dorsal rim area of the compound eye. Microsc Res 40. Johnsen S, et al. (2006) Crepuscular and nocturnal illumination and its effects on color Tech 47(6):368–379. perception by the nocturnal hawkmoth Deilephila elpenor. J Exp Biol 209(Pt 5): 20. Sauman I, et al. (2005) Connecting the navigational clock to sun compass input in 789–800. monarch butterfly brain. Neuron 46(3):457–467. 41. Batschelet E (1981) Circular Statistics in Biology (Academic, London). 21. Weir PT, Dickinson MH (2012) Flying Drosophila orient to sky polarization. Curr Biol 42. Labhart T (1996) How polarization-sensitive interneurones of crickets perform at low 22(1):21–27. degrees of polarization. J Exp Biol 199(Pt 7):1467–1475.

11400 | www.pnas.org/cgi/doi/10.1073/pnas.1501272112 el Jundi et al. Downloaded by guest on October 1, 2021