Despite their small brains, insects can navigate over long distances by orienting using visual landmarks [], skylight polarization [], and sun position []. Although Drosophila are not generally renowned for their navigational abilities, mark-and-recapture experiments in Death Valley revealed that they can fly nearly 15 km in a single evening []. To accomplish such feats on available energy reserves [], flies would have to maintain relatively straight headings, relying on celestial cues []. Cues such as sun position and polarized light are likely integrated throughout the sensory-motor pathway [], including the highly conserved central complex []. Recently, a group of Drosophila central complex cells (E-PG neurons) have been shown to function as an internal compass [], similar to mammalian head-direction cells []. Using an array of genetic tools, we set out to test whether flies can navigate using the sun and to identify the role of E-PG cells in this behavior. Using a flight simulator, we found that Drosophila adopt arbitrary headings with respect to a simulated sun, thus performing menotaxis, and individuals remember their heading preference between successive flights—even over several hours. Imaging experiments performed on flying animals revealed that the E-PG cells track sun stimulus motion. When these neurons are silenced, flies no longer adopt and maintain arbitrary headings relative to the sun stimulus but instead exhibit frontal phototaxis. Thus, without the compass system, flies lose the ability to execute menotaxis and revert to a simpler, reflexive behavior.

Results

4 el Jundi B.

Warrant E.J.

Byrne M.J.

Khaldy L.

Baird E.

Smolka J.

Dacke M. Neural coding underlying the cue preference for celestial orientation. 15 Pegel U.

Pfeiffer K.

Homberg U. Integration of celestial compass cues in the central complex of the locust brain. 16 Heinze S.

Reppert S.M. Sun compass integration of skylight cues in migratory monarch butterflies. 12 Götz K.G. Course-control, metabolism and wing interference during ultralong tethered flight in Drosophila melanogaster. 21 Hoyer S.C.

Eckart A.

Herrel A.

Zars T.

Fischer S.A.

Hardie S.L.

Heisenberg M. Octopamine in male aggression of Drosophila. 22 Reichardt W.

Poggio T. Visual control of orientation behaviour in the fly. Part I. A quantitative analysis. 23 Maimon G.

Straw A.D.

Dickinson M.H. A simple vision-based algorithm for decision making in flying Drosophila. 8 Warren T.L.

Weir P.T.

Dickinson M.H. Flying Drosophila melanogaster maintain arbitrary but stable headings relative to the angle of polarized light. 9 Weir P.T.

Dickinson M.H. Flying Drosophila orient to sky polarization. 24 Wolf R.

Gebhardt B.

Gademann R.

Heisenberg M. Polarization sensitivity of course control in Drosophila melanogaster. 25 el Jundi B.

Foster J.J.

Khaldy L.

Byrne M.J.

Dacke M.

Baird E. A snapshot-based mechanism for celestial orientation. 15 Pegel U.

Pfeiffer K.

Homberg U. Integration of celestial compass cues in the central complex of the locust brain. Figure 1 Flies Navigate Using a Sun Stimulus and Retain Memory of Their Heading Show full caption (A) A tethered fly, backlit with infrared light, is surrounded by a cylindrical LED display; a single 2.4° spot simulates the sun. (B) Example trace showing closed-loop behavior. After ∼90 s, the fly stabilized the sun stimulus at a heading of −92° (dashed red line). (C) Heading during a stripe presentation. (D) Polar representation of data for flies presented with a sun stimulus, with a stripe, and in the dark. Angular position indicates a fly’s mean heading; radial distance indicates vector strength. Red line indicates population mean heading with a circular 95% confidence interval; a histogram of mean headings is plotted around each circle. Due to high variance, we could not calculate a 95% confidence interval for the dark data and do not present a population mean heading, as it is not meaningful with such low vector strengths. A Rayleigh test indicates that headings in the dark are uniformly distributed (p = 0.158), whereas this test rejects uniformity for all datasets with visual feedback throughout the study (p < 0.008 in all cases). (E) Sun versus stripe headings for data shown in (D). Data are repeated on the vertical axis to indicate their circular nature. Diagonal line indicates identical heading over both trials. Error bars indicate circular variance multiplied by an arbitrary scale factor, 36, for visibility. Distributions of mean headings for the sun and stripe are different (Mardia-Watson-Wheeler, W = 13.916, p = 0.001). (F) Heading in first trial plotted against second trial heading for increasing inter-trial intervals; plotting conventions are as in (E). Only flies for which both trials had a vector strength >0.2 are plotted. The black lines again indicate exact correspondence between the first and second sun flight headings, which we refer to as the fixed memory (FM) model. Blue lines indicate the expected shift in headings if flies performed a full time compensation (TC) model, assuming a sun movement of 15° hr−1. (G) Headings for first and second sun presentations for flies in which both trials had a vector strength greater than 0.2; plotting conventions are as in (D). Note that the first sun trial for the 5-min dataset is the same data as in (D), except with the 0.2 vector strength cutoff applied. The bottom row of plots indicates the change in heading, with black and blue lines indicating expected values for FM and TC models, respectively. (H) Distribution of 10,000 bootstrapped heading differences between random pairings of first and second trials from (F). Red line indicates mean heading difference of observed data; p value, proportion of resampled differences that are smaller than the observed mean heading difference. (I) Statistical comparison of FM and TC models. Gray histogram shows the distribution of the difference in mean squared residuals (ΔMSR) between the TC and FM models calculated from 10,000 random samples of 20 data pairs. Blue shading and p values indicate the proportion of subsampled ΔMSRs in which the TC model performed better than the FM model. To follow a straight course, animals must maintain a constant heading relative to a fixed, distant landmark, a strategy termed “menotaxis.” We tested the hypothesis that Drosophila can perform menotaxis using the sun by placing tethered wild-type female flies in a flight simulator and presenting an ersatz sun stimulus ( Figure 1 A). The fly was surrounded by an array of LEDs, on which we presented either a single 2.4° bright spot on a dark background or a 15°-wide dark vertical stripe on a bright background. Given previous studies on other species [], we expected that flies would react to our small bright spot as they would to the actual sun; thus, we called it a “sun stimulus.” Experiments were conducted in closed loop, so that the difference in stroke amplitude between the fly’s two wings determined the angular velocity of the stimulus []. Flies generally maintained the dark stripe in front of them ( Figures 1 C and 1D), a well-characterized behavior termed “stripe fixation” []. However, when presented with the sun stimulus, individual flies adopted arbitrary headings, thus exhibiting menotaxis ( Figures 1 B and 1D). We quantified how well flies maintained a heading by calculating vector strength, which is the magnitude of the mean of all instantaneous unit heading vectors for the entire flight. A vector strength of 1 would indicate that a fly held the stimulus at the exact same heading during the entire flight bout. Because we tested each individual with both a stripe and sun stimulus, we could compare the flies’ performance under the two conditions. We found no correlation between the mean heading exhibited by individual flies during sun menotaxis and stripe fixation (p = 0.143; see STAR Methods for details; Figure 1 E), suggesting that heading preference for the sun stimulus is independent of the response to a vertical stripe. To ensure that flies’ stabilization of the sun stimulus was not an artifact of our feedback system, we also conducted control closed-loop experiments, in which the bright spot was switched off. As expected, the flies exhibited no orientation behavior under this condition, with all vector strength values lower than 0.16 ( Figure 1 D). Collectively, these experiments indicate that flies are capable of orienting to a small bright spot and that this behavior is distinct from stripe fixation. Drosophila can also perform menotaxis using the axis of linearly polarized light []. It is not known whether the orientation responses of flies to the sun and polarized light are independent, as they are in dung beetles [], or linked to create a matched filter of the sky, as they are in locusts [].

8 Warren T.L.

Weir P.T.

Dickinson M.H. Flying Drosophila melanogaster maintain arbitrary but stable headings relative to the angle of polarized light. Given that individual flies adopt arbitrary headings with respect to the sun stimulus, we next tested whether they retain a memory of their orientation preference from one flight to the next. We presented flies with the sun stimulus in closed-loop, interrupted flight for a defined interval (5 min, 1 hr, 2 hr, or 6 hr) and then again presented the sun stimulus. To provide an independent metric of flight performance, we also presented a stripe under closed-loop conditions for 1 min before the first sun bout and after the second. In all cases, distributions of mean headings for the sun and stripe trials were different (Mardia-Watson-Wheeler test: 5 min, W = 13.916, p = 0.001; 1 hr, W = 12.551, p = 0.002; 2 hr, W = 28.891, p = 0.000; 6 hr, W = 10.256, p = 0.006), and these results were robust to excluding any trials with vector strengths less than 0.2 (all ps < 0.03). Across inter-flight intervals of 5 min, 1 hr, 2 hr, and even 6 hr, flies tended to remain loyal to their first heading during the second flight ( Figures 1 F and 1G). If each fly adopted the identical heading in both flights, the mean heading difference would be zero, whereas if there was no correlation in heading from one flight to the next, the mean absolute value of the heading differences would be 90°, provided that the orientations were uniformly distributed. To test whether the consistency in flight-to-flight orientation could arise from chance, we iteratively shuffled pairings of mean heading values of first and second flights 10,000 times and compared the resulting bootstrapped distributions with the mean absolute heading difference of the actual data ( Figure 1 H). In all cases, the measured mean difference was substantially less than the mean of the bootstrapped values (5 min: 49.8° versus 76.3°; 1 hr: 61.0° versus 77.4°; 2 hr: 62.8° versus 83.8°; 6 hr: 61.4° versus 74.4°). We calculated probability values directly from the proportion of simulations that resulted in a smaller mean absolute angle difference than the observed data ( Figure 1 H). In all cases, this probability was quite low (5 min: p = 0.000; 1 hr: p = 0.011; 2 hr: p = 0.002; 6 hr, p = 0.029). Because the heading value of an individual trial is unreliable when vector strength is low, this analysis was conducted after excluding trials in which the vector strength was less than 0.2 (36% of all trials). However, the probabilities calculated using the entire dataset were also very low, except for the 6-hr gap (5 min: p = 0.000; 1 hr: p = 0.028; 2 hr: p = 0.001; 6 hr: p = 0.084). Collectively, these results suggest that headings are not selected at random with each subsequent takeoff but, rather, that flies remember their headings from previous flights for up to 2 hr and possibly longer. A similar result was found for the orientation responses to linearly polarized light, although only a 5-min time gap was tested []. Fully determining the mechanisms by which flies attain their initial heading preference (i.e., genetic versus developmental versus learning) requires experiments that are beyond the scope of this study.

−1 [ 26 Blewitt M. Celestial Navigation for Yachtsmen. Monarch butterflies and bees, which use a sun compass for migration and foraging, respectively, exhibit time compensation so that their orientation preference adjusts for the azimuthal motion of the sun through the sky. The time course of our two-flight experiments allowed us to test whether flies also utilize a time-compensated sun compass. The blue diagonal lines in Figure 1 F plot the prediction of a time-compensated (TC) model, assuming a sun procession of 15° hr], whereas the black lines indicate the prediction of a fixed memory (FM) model in which the headings of the first and second flights are identical. Visual inspection suggests that the FM model predicts the data distributions better than the TC model. This impression was confirmed by a statistical analysis in which we randomly resampled 20 values 10,000 times, in each case calculating the difference in the mean square residual (MSR) values for the TC and FM models ( Figure 1 I). We then used the bootstrapped distributions to determine the probability that the TC model predicted the data better than the FM model (5 min: p = 0.484; 1 hr: p = 0.563; 2 hr: p = 0.173; 6 hr, p = 0.020). As expected, the two models do equally well over short duration gaps, but as the gap between flights increases, the TC model does increasingly worse in predicting the relationship between the headings of the first and second flights. Thus, our experiments suggest that Drosophila do not compensate their sky compass to adjust for the azimuthal motion of the sun.

27 Rieger D.

Fraunholz C.

Popp J.

Bichler D.

Dittmann R.

Helfrich-Förster C. The fruit fly Drosophila melanogaster favors dim light and times its activity peaks to early dawn and late dusk. The finding that flies remember their flight heading for at least 2 hr makes ethological sense. Drosophila are crepuscular, exhibiting dawn and dusk activity peaks []. Assuming that our laboratory measurements are representative of dispersal events, a memory that allows an individual to fly straight for a few hours would be sufficient to bias a day’s migration in one direction. To our knowledge, there is no evidence that Drosophila make multi-day, long-distance migrations that would require the ability to maintain a constant course from one day to the next or a TC sun compass. The most parsimonious ecological interpretation of their sun orientation behavior is that it allows flies to disperse opportunistically to new sources of food and oviposition sites within a single day.

17 Seelig J.D.

Jayaraman V. Neural dynamics for landmark orientation and angular path integration. 18 Turner-Evans D.B.

Wegener S.

Rouault H.

Franconville R.

Wolff T.

Seelig J.D.

Druckmann S.

Jayaraman V. Angular velocity integration in a fly heading circuit. 19 Green J.

Adachi A.

Shah K.K.

Hirokawa J.D.

Magani P.S.

Maimon G. A neural circuit architecture for angular integration in Drosophila. 28 Wolff T.

Iyer N.A.

Rubin G.M. Neuroarchitecture and neuroanatomy of the Drosophila central complex: A GAL4-based dissection of protocerebral bridge neurons and circuits. 17 Seelig J.D.

Jayaraman V. Neural dynamics for landmark orientation and angular path integration. 18 Turner-Evans D.B.

Wegener S.

Rouault H.

Franconville R.

Wolff T.

Seelig J.D.

Druckmann S.

Jayaraman V. Angular velocity integration in a fly heading circuit. 19 Green J.

Adachi A.

Shah K.K.

Hirokawa J.D.

Magani P.S.

Maimon G. A neural circuit architecture for angular integration in Drosophila. 29 Kim S.S.

Rouault H.

Druckmann S.

Jayaraman V. Ring attractor dynamics in the Drosophila central brain. 29 Kim S.S.

Rouault H.

Druckmann S.

Jayaraman V. Ring attractor dynamics in the Drosophila central brain. 17 Seelig J.D.

Jayaraman V. Neural dynamics for landmark orientation and angular path integration. 28 Wolff T.

Iyer N.A.

Rubin G.M. Neuroarchitecture and neuroanatomy of the Drosophila central complex: A GAL4-based dissection of protocerebral bridge neurons and circuits. 29 Kim S.S.

Rouault H.

Druckmann S.

Jayaraman V. Ring attractor dynamics in the Drosophila central brain. Figure 2 E-PG Neuron Activity Correlates with Both Sun and Stripe Positions Show full caption (A) Ca2+ imaging schematic. (B) Glomeruli assignment in protocerebral bridge based on an SD of GCaMP6f fluorescence in E-PG terminals. (C) Continuous circular representation of angular position based on glomeruli positions in (B). 29 Kim S.S.

Rouault H.

Druckmann S.

Jayaraman V. Ring attractor dynamics in the Drosophila central brain. (D) Example trial from functional imaging experiment. GCaMP6f fluorescence (ΔF/F), shown in grayscale, is plotted in each glomerular position (from C) during 45 s of a sun stimulus presentation. Azimuthal position of the E-PG activity bump (blue trace, computed as in []) and sun position (red trace) co-vary. Histograms showing angular distributions for each trace are shown at the right, as are regressions plotting sun position against bump position. The dark gray regions in the regression plots indicate the sectors of the arena in which the stimulus was not visible to the fly. (E) Same as in (D), but showing data from the second sun presentation. (F) Same as in (D), but showing data from the stripe presentation. (G) Heading during the second sun trial plotted against heading in the first sun trial, with a minimum 5-min inter-trial interval (n = 20; plotted as in Figure 1 E). (H) Polar representation of second sun-bout headings, plotted as in Figure 1 D. Shaded area indicates sector that was not visible to fly. (I) E-PG bump-to-stimulus offset for the second sun trial plotted against the mean sun heading. (J) Regression of the median bump-to-stimulus offset for the second sun trial plotted against the offset for the first sun trial. (K) Bump-to-stimulus offset for stripe plotted against the offset for the sun. See also Videos S1 and S2 The visual information conveying sun position likely provides inputs to the recently identified neurons constituting the fly’s internal compass []. These columnar neurons receive input in the ellipsoid body and send divergent output to the protocerebral bridge and gall, and they are, hence, named E-PG neurons []. These neurons track the azimuthal position of vertical stripes and more complex visual stimuli and, in the absence of visual input, can continue to track azimuthal orientation by integrating estimates of angular velocity []. Given these functional attributes, two obvious questions are whether E-PG neurons respond to a sun stimulus and whether they exhibit different responses to other visual stimuli. We used the split-GAL4 line SS00096 [], which expresses in the E-PG neurons, to drive the genetically encoded calcium indicator GCaMP6f and measured activity in tethered, flying flies using a 2-photon microscope ( Figure 2 A). As described previously, the E-PG neurons tile the toroidally shaped ellipsoid body. Notably, a region of activity, or “bump,” rotates around the ellipsoid body corresponding to azimuthal position ([ Videos S1 and S2 ). Instead of recording from the ellipsoid body, we imaged the activity at E-PG terminals in the protocerebral bridge ( Figure 2 B), because fluorescence signals were stronger in these more superficial glomeruli. Based on well-established anatomy, we re-mapped the neural activity in the medial 16 glomeruli of the protocerebral bridge into the circular reference frame of the ellipsoid body ( Figure 2 C; []) and computed a neural activity vector average, or bump position, for each image (similar to []; see STAR Methods for details).

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eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiI3NWE5MjI1MzY5NjkyNmNmNzE2YjViNzkyMTliNDE4ZCIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjAxMDk2MDU1fQ.CGunIyBQ1MT4XDEBNGDc8EVdboOtaGuVc0oOZ8SMIKgiul7B-azkzqh_zgPbFX5a6dE4xWvwdZAetQWin1OigQQ6QInpZXxG2GBPaL-dXORyIoM85KSy-tmEWJengiAWV_yBclkIunkJpIr2b2JCyPreiLbZTUlWv42aprJLXAA1K8ASr5UmnvkX7x8ltdZz82OOUzY0JJ83Es4INTkljMTNQ1K9YN0MU_Sf7vX1oUpwMLOnt3EdCL2U4J5QxM6iSuaRikf6FMIvcYRm2qbGB27u2P_G9oBORw4-WMD11YzNKsQKdN0zMiQCbJu0w1G8VNJE-FjV7oQGSg4qFKn8mw

17 Seelig J.D.

Jayaraman V. Neural dynamics for landmark orientation and angular path integration. As in our flight arena experiments ( Figure 1 A), flies adopted arbitrary headings with respect to the sun stimulus ( Figures 2 G and 2H), which they maintained over a 5-min break ( Figure 2 G). By presenting sun and stripe stimuli to the same fly, we tested whether these two stimulus types are represented differently by the E-PG neurons. Bump position faithfully tracked the position of both the sun and stripe stimuli ( Figures 2 D–2F). Prior studies found that, while the E-PG bump tracks the azimuthal position of a vertical stripe, it does so with an arbitrary azimuthal angular offset []. We found an identical result with the sun stimulus; the bump rotated with changes in sun position but with a bump-to-stimulus offset that varied from individual to individual. In addition, the bump-to-stimulus offset did not differ between the first and second sun presentation trials or between the sun and stripe presentation trials ( Figures 2 J and 2K). The offset was not correlated with the azimuthal angle at which individual flies tended to hold the sun ( Figure 2 I). Together, these imaging results suggest that the representation of the sun and stripe in the E-PG neurons is similar, despite the distinct behavioral responses to the stimuli, and that the bump-to-stimulus offset does not encode heading preference.