Abstract
To follow a straight course, animals must maintain a constant heading relative to a fixed, distant landmark, a strategy termed menotaxis. In experiments using a flight simulator, we found that Drosophila adopt arbitrary headings with respect to a simulated sun, and individuals remember their heading preference between successive flights—even over gaps lasting several hours. Imaging experiments revealed that a class of neurons within the central complex, which have been previously shown to act as an internal compass, track the azimuthal motion of a sun stimulus. When these neurons are silenced, flies no longer adopt and maintain arbitrary headings, but instead exhibit frontal phototaxis. Thus, without the compass system, flies lose the ability to execute menotaxis and revert to a simpler, reflexive behavior.
One sentence summary Silencing the compass neurons in the central complex of Drosophila eliminates sun navigation but leaves phototaxis intact.
Despite their small brains, insects can navigate over long distances – in some cases, thousands of kilometers – by orienting to sensory cues such as visual landmarks (1), skylight polarization (2–9) and the position of the sun (3, 4, 6, 10). 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 across open desert over the course of a single evening (11). To accomplish such feats on available energy reserves (12), flies would have to maintain relatively straight headings and rely on celestial cues to do so (13).
Celestial cues such as sun position and polarized light are thought to be integrated in the central complex, a set of highly conserved unpaired neuropils in the central brain of arthropods (14). Central complex neurons in locusts (15), dung beetles (4), and monarch butterflies (16) respond to the angle of polarized light and the position of small bright objects mimicking the sun or moon. Extracellular recordings from the central complex in cockroaches revealed neurons that act as head-direction cells in the absence of visual cues or relative to a visual landmark (17). Recently, a group of cells (E-PG neurons) in the Drosophila central complex have been shown to function as an internal compass (18–20), similar to head-direction cells in mammals (21). Using the wide array of genetic tools available to measure and manipulate cell function in Drosophila, we set out to test whether flies can navigate using the sun and to identify the role of E-PG cells in this behavior.
We tested the hypothesis that Drosophila can use the sun to navigate by placing tethered wild-type female flies in a flight simulator and presenting an ersatz sun stimulus (Fig.1A). The fly was surrounded by an array of LEDs on which we presented either a single 2.3° bright spot on a dark background or a 15°-wide dark vertical stripe on a bright background. Given previous studies on other species (4, 15, 16), we expect that flies react to our small bright spot as they would to the actual sun, and thus we call it a ‘sun stimulus’. Experiments were conducted in closed loop, such that the difference in stroke amplitude between the fly’s two wings determined the angular velocity of the stimulus (12). Flies generally maintained the dark stripe in front of them (Fig. 1C, D), a well-characterized behavior termed stripe fixation (22–24). However, when presented with the sun stimulus, individual flies adopted arbitrary headings, thus exhibiting menotaxis (Fig. 1B, D). 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 (Fig. 1E), 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 (Fig. 1D). 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 (8, 9, 25). It is not known whether the orientation responses of flies to the sun and polarized light are independent, as they are in dung beetles (4), or linked to create a matched filter of the sky, as they are in locusts (15).
Given that individual flies adopted arbitrary headings with respect to the sun stimulus, we tested whether they retained 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, 2, or 6 hours), 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. Across inter-flight intervals of 5 minutes, 1 hours, and 2 hours, flies remained loyal to their first heading during the second flight (Fig. 1F). 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 difference 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 bootstrapped 10,000 random pairs of mean heading values from the first and second flights and compared the resulting distribution with the mean absolute heading difference of the actual data (Figure 1G). In all cases, the measured mean difference was less than that of the bootstrapped values (5 min: 54.2° vs. 79.2°; 1 hour: 66.6° vs. 77.4°; 2 hours: 66.8° vs. 84.8°; 6 hours: 71.0° vs. 75.5°). We calculated probability values directly from the proportion of the 10,000 bootstrapped simulations that resulted in a smaller mean absolute angle difference than the observed data (Fig.1G). With the exception of the 6-hour gap, this probability was quite low (5 min: p=0.00; 1 hour: p=0.03; 2 hours: p=0.001; 6 hours, 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, at least for up to 2 hours. A similar result was found for the orientation responses to linearly polarized light (8). Fully determining the mechanisms by which flies attain their initial heading preference (i.e. genetic vs. developmental vs. learning) require experiments that are beyond the scope of this current study.
The finding that flies remember their flight heading for at least 2 hours makes ethological sense. Drosophila are crepuscular, exhibiting dawn and dusk activity peaks (26). Assuming 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 time-compensated 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.
The visual information conveying sun position likely provides inputs to the recently identified neurons constituting the fly’s internal compass (18–20). These columnar neurons receive input in the ellipsoid body and send divergent output to the protocerebral bridge and gall, and are hence named E-PG neurons (27). These neurons track 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 (18, 20, 28). Given these functional attributes, an obvious question is 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 (28), 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 (Fig. 2A). As described previously, the set of 16 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 (18, Movies S1, S2). Instead of recording from the ellipsoid body, we imaged the activity at E-PG terminals in the protocerebral bridge (Fig. 2B) 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 azimuthal space (Fig. 2C, 27) and computed a neural activity vector average, or bump position, for each image (similar to 28; see Materials and Methods for details).
As in our flight arena experiments (Fig 1A), flies adopted arbitrary headings with respect to the sun stimulus (Fig. 2G, H), which they maintained over a 5-minute break (Fig 2G). 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 (Fig. 2D-F). 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 (18). 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 trails (Fig 2J, K). The offset was not correlated with the azimuthal angle at which individual flies tended to hold the sun (Fig. 2I). 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.
We next tested the causal contributions of E-PG neurons to sun navigation and stripe fixation, predicting that the highly variable headings adopted in sun navigation might require the instantaneous positional information provided by E-PG neurons. We took advantage of the sparse expression patterns of three different split-GAL4 lines (Fig. 3A) to selectively drive the inwardly rectifying potassium channel Kir2.1 (29). As a control, we crossed UAS-Kir2.1 to an engineered split-GAL4 line that was genetically identical to the experimental driver lines, but carried empty vectors of the two GAL4 domains in the two insertion sites (30). Driving Kir2.1 in three, separate split-GAL4 lines yielded flies that lost the ability to maintain the sun at arbitrary azimuthal positions, although they could fixate the sun and stripe frontally. To assess the degree to which this effect could have occurred by chance, we employed a bootstrapping approach similar to that used in our time gap experiments. We randomly selected 50 values from our control dataset 10,000 times, in each case calculating the circular variance of the subsampled population. We then determined the proportion of bootstrapped mean variances that had smaller values than the variance of the actual experimental data and concluded that the observed frontal distributions of our experimental groups were highly unlikely to have occurred by chance (SS00096: p=0.000; SS00408: p=0.000; SS00131: p=0.004). Thus, E-PG neuron activity appears necessary for menotaxis, i.e. maintaining the sun in arbitrary non-frontal positions. To our knowledge, this is the first behavioral deficit elicited via experimental manipulation of the compass cell network.
In the absence of normal E-PG function, flies might directly orient toward the sun because they lack the ability to compare their instantaneous heading to a stored value of their directional preference. Such a loss of function in the compass network might unmask a simpler reflexive behavior – phototaxis – that does not require the elaborate circuitry of the central complex. Consistent with this hypothesis, stripe fixation was not different between control and experimental animals. This interpretation is compatible with a recent model that showed frontal object fixation could result from a simple circuit involving two asymmetric wide-field motion integrators, without the need for the central complex (31).
Our findings are consistent with an emerging model of a navigational circuit involving the central complex. E-PG cells have an excitatory relationship with another cell class in the central complex (protocerebral bridge-ellipsoid body-noduli or P-EN neurons), creating an angular velocity integrator that allows a fly to maintain its heading in the absence of visual landmarks (19, 20). Furthermore, the E-PG neurons are homologous to the CL1 neurons described in locusts (32), monarchs (16), dung beetles (4), and bees (33) and likely serve similar functions across taxa. In an anatomy-based model of path integration in bees, CL1 neurons are part of a columnar circuit that provides instantaneous heading information to an array of self-excitatory networks that also receive convergent optic flow information, thereby storing a memory of distance traveled in each direction (33). This information is then retrieved as an animal returns home by driving appropriate steering commands in other classes of central complex neurons. The putative memory cells suggested by this model, CPU4 cells, could be homologous to protocerebral bridge-fan shaped body-noduli (P-FN) neurons described for Drosophila (27). Furthermore, cells responsive to progressive optic flow are found throughout the central complex of flies, including neuropil in the fan-shaped body containing the P-FN cells (34). The authors of the recent path integration model suggest that the CPU4 network in bees might also function to store the desired heading during sun navigation (33). Although our results do not directly test this model, they are consistent with the role of CL1 neurons in providing heading direction to circuits that generate steering commands towards an arbitrary orientation whose memory is stored in the network of CPU4 (P-FN) neurons.
Stripe fixation and sun navigation behaviors may represent two different flight modes in Drosophila. Stripe fixation is thought to be a short-range behavioral reflex to orient towards near objects (12), which in free flight is quickly terminated by collision avoidance (13) or landing behaviors (35). In contrast, navigation using the sun is likely a component of long-distance dispersal behavior that could be used in conjunction with polarization vision (8, 9) either in a hierarchical (4) or integrative (36) manner.
Individuals could differ in where they lie on the continuum of long-range dispersal to local search, which could explain the inter-individual variation we observed in heading fidelity during sun orientation experiments. In general, dispersal is a condition-dependent behavior that is known to vary with hunger or other internal factors (37).
Given the architectural similarity of the central complex among species (14), the celestial compass we have identified in Drosophila is likely one module within a conserved behavioral toolkit (13) allowing orientation and flight over long distances by integrating skylight polarization, the position of the sun or moon, and other visual cues. An independent study has recently found that the E-PG compass neurons are also necessary in walking flies for maintaining arbitrary headings relative to a small bright object (38). The expanding array of genetic tools developed for flies as well as the rapid growth in our understanding of the neural circuitry involved in orientation during walking (18–20) and flight (28) make this a promising system for exploring such essential and highly conserved behaviors.
Acknowledgments
We wish to thank Tanya Wolff and Gerry Rubin for generously providing us with the split-GAL4 lines SS00131 and SS00408 prior to the publication of their manuscript describing them. Crystal Liang and Aisling Murran provided valuable assistance with data collection. Funding: This work was funded by grants from the NSF (IOS 1547918), NIH (U19NS104655), and the Simons Foundation (71582123) to MHD, as well as an NIH NRSA postdoctoral fellowship (F32GM109777) to YMG. Author contributions: PTW and TLW were involved in early experimental design and analysis. YMG, KJL, IKR and MHD conceived of and conducted experiments. YMG characterized sun compass behavior (Fig. 1), IKR conducted functional imaging experiments (Fig. 2), and YMG and KJL performed genetic silencing experiments (Fig. 3). YMG, KJL, IKR and MHD wrote the paper. All authors contributed in editing the final manuscript. Competing interests: Authors declare no competing interests. Data and materials availability: All data will be made available on Dryad upon publication.