Preferred viewing directions of bumblebees (Bombus terrestris L.) when learning and approaching their nest site

1 2 3 4 5 6 7 8 9 1 11 12 13 14 15 16 17 18 19 2 21 22 23 24 25 26 27 28 29 3 31 Preferred viewing directions of bumblebees (Bombus terrestris L.) when learning and approaching their nest site Natalie Hempel de Ibarra 1,3 Andrew Philippides 2, Olena Riabinina 2, Thomas S. Collett 1 Department of Biology and Environmental Science, School of Life Sciences 1 and Department of Informatics, School of Science and Technology 2, University of Sussex Present address 3 : Department of Psychology, University of Exeter Keywords: Bumblebees, navigation, landmarks, learning, orientation flights Summary Many bees and wasps learn about the immediate surroundings of their nest during learning flights, during which they look back towards the nest. The acquired visual information then guides their subsequent returns. Visual guidance is simplified by their adoption of similar compass directions during learning and return flights. To understand better the factors determining the particular viewing directions that insects choose, we have recorded the learning and return flights of a ground-nesting bumblebee in two visual environments - a flat roof with an open panorama and an enclosed garden with a partly open view between north and west. In both places, bees left and returned to an inconspicuous nest-hole in the centre of a tabletop, with the hole marked by one or more nearby cylinders. On the open roof, the bees predominant facing direction switched over the course of the day. Bees faced south in late morning and early afternoon. They faced north, or had no clear preference later on. In the partly enclosed garden, bees tended to face north throughout the day. When white curtains, which distributed skylight more evenly, were placed around the table in the garden, bees faced both north and south. In general, bees faced predominantly along a north south axis, choosing the pole in the brighter hemisphere. The nearby cylinders had a minor influence on viewing direction. This 1

32 33 34 behaviour may be a compromise between maintaining a single viewing direction for efficient view-based navigation, and facing the brighter hemisphere to aid detection of landmarks seen against the ground. 2

35 36 37 38 39 4 41 42 43 44 45 46 47 48 49 5 51 52 53 54 55 56 57 58 59 6 61 62 63 64 Introduction Many bees and wasps learn the relationship between visual landmarks and a nest or feeding site during elaborate learning flights that they perform on their first few departures from the site (Bates, 1863; Wagner, 197; Wolf, 1926; Wolf, 1927; Opfinge, 1931; Tinbergen 1932; Becker, 1958; van Iersel and van der Assem, 1964; Lehrer, 1991; Collett and Lehrer, 1993; Zeil, 1993 a,b). The general interest of these flights is that they present a rare example of a relatively stereotyped manoeuvre that has evolved for acquiring information about visual landmarks. The information is used primarily for just one task that of guiding the insect s return to the site that it is leaving. Learning flights should therefore be structured so that the stored information is in a suitable format for guiding the returns. Two notable features of the flights play an obvious role in simplifying visually guided homing. First, over much of the flight, the insect faces in the rough direction of the site that it is leaving (reviewed in Wehner 1981). Second, the azimuth of the preferred orientation of the insect s longitudinal body axis, when it is close to the site, is similar across multiple learning and return flights (Zeil 1993a,b; Collett 1995). The preferred orientation that insects choose seems to differ between species. The solitary ground nesting wasp, Cerceris sp. On emerging from a nest-hole with a small cylinder close to it backs away so that the cylinder is always seen beyond the nest (Zeil, 1993a). The wasp s orientation is roughly parallel to the vector from the nest to the nearby landmark. On its return to the nest-hole, the wasp approaches the nest, facing in the same orientation as it did on departure (Zeil, 1993b). The cylinder is again seen on the far side of the nest and can be used as a guiding beacon throughout the approach trajectory. Social wasps and honeybees learning about a food site on departure also have a preferred body orientation that is common to their departing and return flights, but the orientation of these wasps (Collett, 1995; Collett and Rees, 1997) and bees (Collett and Baron, 1994) is influenced less strongly by the position of a nearby landmark, and in an insect s final approach to the nest, the landmark often cannot act as a beacon. 3

65 66 67 68 69 7 71 72 73 74 75 76 77 78 79 8 81 82 83 84 85 86 87 88 89 9 91 92 93 94 95 We ask here what factors might determine the horizontal orientation of the body axis of the bumblebee, Bombus terrestris L., when learning and finding its nest site and whether its preferred orientation might be related to the visual ecology of its habitat. Bombus terristris is a good species for such a study because it is relatively easy to record flights from many bees in similar conditions. Also, because it nests in holes in the ground and normally emerges onto a horizontal surface, it is straightforward to examine how compass cues and local landmarks influence the bee s facing direction immediately after it leaves the nest. The entrance to a bumblebee s nest is often hidden by ground cover. When approaching its nest, a bee gradually loses height so that small, upright landmarks close to the entrance, like plants or stones, are first seen against ground cover, rather than against the sky. A simple example of this arrangement of landmark and background is shown in Figure 1, where an upright branch is viewed against grass with the sun at different angles of azimuth relative to the viewing direction of the camera. When the camera faces the sun, the vertical branch contrasts darkly against grass, and the shadow of the branch is at a lower elevation than the branch itself. With the sun behind the camera, the contrast difference between branch and grass is much smaller. The shadow is dark and lies at a similar elevation to the branch and could easily be mistaken for it. Thus, the branch will be most visible against the background and also most easily distinguished from cast shadows when the bee faces the sun. The branch has high contrast over at least 6 o either side of the sun s azimuth (Fig. 1), so that viewing angle is not critical. It could therefore be helpful in detecting landmarks for bees to face towards the sun during learning and return flights. But there may be other competing constraints. Since bumblebees probably have a sizeable blind area behind them, the selected orientation may be chosen according to the arrangement of landmarks near the nest to give an approaching bee guidance cues within its frontal visual field. Second, since landmark guidance is likely to be view-based and dependent on retinotopic memories, there is an advantage to sticking to one direction of flight and a single horizontal body orientation. The use of that strategy keeps down the number of memories that need be stored. Also, a 4

96 97 98 99 1 11 12 13 14 15 16 17 18 19 11 111 112 113 114 115 116 117 118 119 12 121 122 123 124 125 126 foraging trip can take as much as two hours so that adjacent learning and return flights can occur with the sun in distinctly different positions. It might thus be disadvantageous for the bee to change its orientation continuously to track the sun. Nonetheless, it may help in recognizing landmarks to keep the sun roughly in the frontal hemisphere of the eye by means of occasional switches of body orientation over the day. We have recorded learning and return flights in two different visual environments using a variety of landmark arrangements. Most data were obtained from bees tested in a domestic garden in Lewes, East Sussex UK. The garden is partway up the south side of a valley with steep southern flanks that for much of the morning occluded the sun. The house is built into the hill and it obscured afternoon sun from the south west. The horizon is lowest in a roughly 8 o sector between north and west. The second set of recordings was made on a more open site on the flat roof of a building at the University of Exeter. The view from the roof is clear to the south, but a little broken by trees and other buildings to the north. To see whether bumblebees behave like Cerceris and orient towards single landmarks, we placed a single landmark roughly north or east or south or west of the nest-hole. To reduce the possibility that landmarks might induce a directional bias we placed four cylinders symmetrically about the nest-hole. Out of curiosity, we also used two cylinders to the west or to the east of the nest-hole. We report here the regularities in body orientation that we found. Methods Flights from and to a small artificial nest-hole in the centre of a tabletop (18 cm by 15 cm in the garden and 15 cm by 15 cm on the roof) were recorded between late June and late August in 27 in the garden and between late May and early June in 28 on the roof. During the latter period, the sky was often clouded over. The tabletop was covered with white bath mats which provided contrast against which the bee could be viewed. The pile of the mat gave visual texture that we found to be essential for bees to 5

127 128 129 13 131 132 133 134 135 136 137 138 139 14 141 142 143 144 145 146 147 148 149 15 151 152 153 154 155 156 157 stabilise their flight when flying low over the substrate. Varying numbers of black cylinders, 2 cm high x1.8 cm wide in the garden and 2 or 8x1.8 cm on the roof were used as landmarks. In the garden, the camcorder (Sony HD, 25 f.p.s, 5 f.p.s. interlaced) was suspended from scaffolding oriented along a WSW and ENE axis 2 m above the table. The orientation of the scaffolding turned out to be oblique to the bees preferred viewing direction so that we doubt it is an important determinant of their orientation. On the roof, a vertical strut at each N, E, S or W corner of the table supported a thin cross 15 cm above the table, to which the camera was fixed. To avoid complications that might be cause by the shadows, we only analysed those flights in the 28 data setin which shadows were absent. A separate tape-deck or second camcorder fed by the camcorder above the nest-hole served as a data recorder. For one experiment in the garden, the scene beyond the table was masked off by encircling the table with a double thickness of 2 m long, white sheeting hung from the scaffolding. Commercially reared colonies of B. terrestris came from Koppert UK. During experiments, a colony nest-box was fixed below the table and its entrance hole was attached by a series of tubes, gates and blind alleys to the hole in the centre of the tabletop through which the bees left their nest and later returned to it. By manipulating gates, bees could usually be persuaded to leave singly. Individual bees were marked with colours and/or number plates. Flights to and from the nest were recorded from above over several days. Each group of about 1 or more bees experienced only one arrangement of landmarks. We rarely obtained complete records of an individual bee s learning and return flights. Data from flights were discarded when returning bees coincided and interfered with departing ones, and sometimes bees returned when the recording device was turned off. Although height was not monitored, observation revealed that the bees fly close to the table for much of each learning flight. Height is only gained at the end of the flight segments that were captured by the camera. 6

158 159 16 161 162 163 164 165 166 167 168 169 17 171 172 173 174 175 176 177 178 179 18 181 182 183 184 185 186 187 188 The information from digital tapes was transferred to a hard drive using Adobe Premiere Pro. Software was written in MatLab to extract a bee s coordinates and the orientation of its body axis. The program made it possible to check the computed values and then, when necessary adjust these values by hand. In all about 693 learning flights and 533 return flights were recorded. In addition to analysing whole flights, we also examined orientations when the bee faced within 1 o of the nest or landmark (i.e. when a line from the centre of the nest-hole or landmark made an angle of 1 o or less with the bee s longitudinal body axis). To analyse statistical differences between the orientations adopted in different experimental conditions, a single measure is needed for each flight. We put the orientations from a single flight, or selected frames of that flight, into 1 o bins and determined the peak orientation, that is the bin with the most counts. Means of the peak orientations, their standard errors and vector lengths were then computed for all flights grouped according to landmark arrangement or time of day. The statistical significance of differences between conditions was assessed using Watson s, non parametric U² test. Statistical analysis of circular data was performed using the software package Oriana. Results We will describe the spatial pattern of the bees learning and return flights elsewhere. Here we concentrate on the preferred orientation of their longitudinal body axis in a horizontal plane. The area surveyed by the camera varied somewhat between landmark arrangements, but typically extended at least 5 cm from the nest in all directions. Within this area bees in the enclosed garden faced predominantly north, but with south and northwest peaks in some experimental conditions. On the more open roof, the bees predominant orientation was south, with a small component to the north. We start by comparing the orientation of the bees body axis during flights recorded in the garden and the roof for two 7

189 19 191 192 193 194 195 196 197 198 199 2 21 22 23 24 25 26 27 28 29 21 211 212 213 214 215 216 217 218 219 arrangements of cylinders: one cylinder roughly to the north of the nest-hole and four cylinders distributed symmetrically about the nest-hole. In the second part of the results, we describe the bees orientation for various landmark arrangements tested in the garden. 1. Comparing flights in the enclosed garden and on the more open roof One cylinder 8 cm north of the nest-hole The orientation of the bees body axis during learning and return flights in the garden is shown in Fig. 2. We wanted to know first what consistency there might be across all the flights of all the bees exposed to one cylinder to the north. Figure 2A shows a histogram of the bee s orientation accumulated over every frame of every flight. Second, to test whether this distribution might be biased by very long flights, we determined the peak orientation for each flight (see Methods) and made a histogram of these values (Fig. 2B). Both distributions show a prominent peak roughly to the north. Third, to examine how the bees orientation varies with distance from the nest, the distribution of orientations is pooled over concentric 2 cm wide annuli centred on the nest and plotted as a false colour map of body orientation against distance (Fig. 2C,D). On learning flights, a broad north peak can be seen to extend to about 4 cm from the nest. There seem to be two hot spots over this distance one close to the nest at about 6 cm and the other at more than 3 cm from the nest, possibly hinting that bees store views at these two distances. Interestingly, the bees seem to face into their preferred orientation further from the nest on return than on learning flights. Perhaps they recognize a stored view and adopt their preferred orientation at some distance from where the view was acquired. But what is indubitable is that all three orientation plots reveal a close correspondence between the bees orientations on learning and return flights. The distributions with a single cylinder to the north are different for flights recorded on the roof (Fig. 3). Here the north peak is much less prominent. Instead, there is a large south peak, which is clearer for learning flights than for return flights (Fig,. 3A, B). The distribution of orientations varies across the day (Fig. 3C, E). Bees faced south around midday and early afternoon and north later on. The mean vector of the peak orientations 8

22 221 222 223 224 225 226 227 228 229 23 231 232 233 234 235 236 237 238 239 24 241 242 243 244 245 246 247 248 249 25 adopted within each learning flight before 15:3 was 148 (n=38, vector length=.3). The mean vector after 15:3 was 357 (n=29, vector length=.48). The two mean vectors differ significantly (Watson s U² test: U² =.388, p<.1). Similar significant differences are found on return flights. The mean vector before 16:3 was 196 (n=21, vector length=.4) and after 16:3 it was 344 (n=21, vector length=.6), with Watson s U² giving U² =.57, p<.1. The time course of the switch from north to south is shown in Fig.3D,F by plotting the number of peak orientations that were within ±45 o of north, east, south and west during successive two-hour intervals. The data are too few to be sure, but they raise the possibility that a switch in orientation might be expressed a little later on return than on learning flights, a pattern that would fit with the long duration of some foraging flights. 4 cylinders surrounding the nest-hole The bees orientation in Figs. 2 and 3 might have been biased by the single landmark to the north. Any such bias was avoided by using four cylinders distributed symmetrically about the nest-hole. Each cylinder was placed 2 cm from the nest-hole, with one at each cardinal compass point. Bees, nonetheless, continued to face north in the garden (Fig. 4A,B). The mean vector of peak orientations of learning flights is 354 (n=3, vector length=.81) and of return flights is 355 (n=27, vector length=.59). To test whether the north facing peak might be a consequence of the relatively low horizon to the northwest, we hung 2 m long white curtains around the experimental table. The curtains both obscured the more distant visual features and distributed the light more evenly. A new group of bees recorded with the curtain in place exhibited a bimodal distribution of body orientations, with a prominent south peak in addition to the north peak (Fig. 4C,D). A statistical indication of bimodality is that the mean vector of the peak orientations of learning flights were longer when computed axially over 18 o (learning flights: mean vector= 166, vector length=.52, n= 38; return flights: mean vector= 174, vector length=.55, n= 43) than when computed normally over 36 o (learning flights: mean vector= 252, vector length=.23, n= 38; return flights: mean vector= 147, vector length=.27, n= 43). 9

251 252 253 254 255 256 257 258 259 26 261 262 263 264 265 266 267 268 269 27 271 272 273 274 275 276 277 278 279 28 281 This bimodal pattern of orientation also occurred at the start of learning flights when bees keep close to the nest-hole. In the orientation distributions of Fig. 4E,F we have only included those frames in which the bee faced the nest-hole and was no further than 7 cm from it (peak orientations of learning flights: axial mean vector= 166, vector length=.51, n= 38; peak values of return flights: axial mean vector= 157, vector length=.27, n= 43). Thus, the bimodality is more likely to be a direct effect of the appearance of the surroundings influencing the bees orientation as soon as they emerge from the nest-hole than an indirect effect of the sheeting at the edge of the table constraining and changing the pattern of the bees flight. The distribution of orientations for flights on the roof is much more variable and exhibits a small south peak (Fig. 5A, B). Again the distribution varies with time of day (Fig.. 5C- F) with the relative frequency of south peaks greatest around midday. The mean vector of the peak orientations of learning flights was 154 (n=117, vector length=.25) before 15:3. That after 15:3 was not well directed (mean=334, n=99, vector length=.7). The two distributions differ significantly (Watson s U² test: U² =.374, p<.2). Similar significant differences are found between return flights. The mean vector before 16:3 was 171 (n=72, vector length=.33) and after 16:3 pm it was 48 (n=65, vector length=.16), and Watson s U² test gave U² =.318, p<.5. From 15: onwards the pattern is unclear. North peaks were more frequent in the late afternoon, but from 18: there is no clear preference. No significant differences were seen between learning and return flights in the morning (U² =.73,.5 > p >.2) or afternoon (U² =.83,.5 > p >.2). This similarity between learning and return flights suggests that the temporal pattern is more than a randomly noisy distribution. To examine whether there may also be changes of orientation with time of day in the garden, we have pooled data from all the cylinder arrangements for which the mean vectors of the flights were within 4 o of north (i.e. all the conditions listed in Tables 1 and 2 except for 2 west). In Fig. 6, we plot the number of peak orientations that were within ±45 o of north, east, south and west for every hour during the day. Throughout the day, 1

282 283 284 285 286 287 288 289 29 291 292 293 294 295 296 297 298 299 3 31 32 33 34 35 36 37 38 39 31 311 312 most peak orientations lay within the north category (learning flights: 142 out of 276; return flights: 178 out of 259). For learning flights, south is the next most populated category (55 out of 259 flights). The pattern differs for return flights. In this case, west is the second most occupied category (5 out of 276 flights) and the proportion of west peaks rises as the day progresses and the sun moves westward. Taken together, the data of this section suggest that the distribution of light in the bees surroundings plays a significant role in setting their preferred orientation. 2. Landmark arrangements and orientation of body axis in the garden We ask in this section whether the arrangement of local landmarks close to the nest-hole influences the bees body orientation. For each landmark arrangement, we pool orientations over all frames of all learning or return flights. But learning flights are likely to consist of several components. There are flight segments towards and facing the nest, in which bees may store views of the nest s surroundings (Collett and Lehrer, 1993), but also landmark based manoeuvres and flight segments away from the nest. Therefore, we also restrict analysis of the bees body orientation to frames in which bees face the nest. 1% of the frames on learning flights are nest facing, and on return flights the proportion is slightly higher, 13%. This difference is consistent across landmark arrangements. We examined the bees orientation during learning and return flights with a single cylinder placed 8 cm and roughly (i.e. about 14 o west of the cardinal directions. See Table 1) north, south, east, or west of the nest-hole. With the cylinder to the north, the peak of the distribution of orientations and the mean vectors of both learning and return flights were slightly to the west of compass north, both when all frames were included and for those frames in which the bees faced the nest (Figs. 2, 7A, Table 1). With the cylinder to the south, east or west, the peak of the distribution of orientations and the mean vector of learning flights pointed approximately north for frames in which bees faced the nest (Fig. 7B-D, Table 1). When all frames were included, the means of the peak orientations of learning and return flights shifted east when the cylinder was to the east and west when it was to the west (Table 1) suggesting the influence of landmark 11

313 314 315 316 317 318 319 32 321 322 323 324 325 326 327 328 329 33 331 332 333 334 335 336 337 338 339 34 341 342 343 based flight patterns. The peaks of the distributions of orientations were not much changed but the flanks were higher to the east or west when the cylinder was to the east or west of the nest respectively (Fig. 7B, D). We experimented with a white curtain behind the cylinder to the west to increase that cylinder s visibility, but with no effect on the bees orientation (data not shown). In all cases, the major peaks of the distributions are narrower for nest facing frames than for distributions including all frames, probably for the reasons given in the previous paragraph. The pattern was much the same for return flights, except when the cylinder was to the south, in which case bees facing the nest had to adjust their orientation to avoid colliding with the cylinder. One notable feature of Fig. 7 is that with all frames included the peaks are narrower on return than on learning flights. On return flights, the bee is primarily aiming for the nest, whereas learning flights are more complex. We also tested a square array of cylinders rotated 55 o west relative to the array shown in Fig. 4. Once again, the major peak of the distribution of orientations was roughly north (Fig. 7F) for learning and return flights, but the flanks were higher to the west of the peak, as is borne out by the mean vectors of peak orientations (Table 2). When the two cylinders to the west of the nest were removed, leaving two cylinders to the east, the distribution was also roughly north (Fig. 7E). The distribution of orientations changed dramatically when two cylinders to the were removed. With only two cylinders to the west of the nest-hole, the peak shifted westward (Fig. 8A, Table 2) to match the direction of the northwest cylinder from the nest. It did so when all frames were included and when the bee faced the nest or the northwest cylinder. The same northwest orientation is evident when orientation is plotted against distance from the nest for frames in which the bee faced the NW cylinder (Fig. 8B). When the bee faced the nest, orientation was roughly northwest until the bee was within 6 cm of the nest-hole at which point the bee started to turn so that it mostly faced in a more westerly direction (Fig. 8C). Perhaps this shift allows a view stored near the nest to include both cylinders so that both can contribute to pinpointing the nest on returns. The fact that the major 12

344 345 346 347 348 349 35 351 352 353 354 355 356 357 358 359 36 361 362 363 364 365 366 367 368 369 37 371 372 373 374 preferred orientation is along the vector from nest to NW cylinder suggests that in addition a view is stored close to the nest when facing the NW cylinder. Body orientations during learning and return flights are remarkably similar when bees face either the nest or the cylinder. Orientations on learning flights seems to be coherent over a longer distance from the nest than they are with a single cylinder (cf. Fig. 2), possibly because two cylinders stabilize the bees flight path better than one. A somewhat perplexing feature of these results is that two cylinders to the east of the nest-hole do not bias the bees orientation eastward relative to their orientation with four cylinders present. A possible reason for this difference is that, unlike the NW cylinder, the NE cylinder does not lie within the low sector of the horizon. Thus, only with two landmarks to the west did bumblebees behave somewhat like the solitary wasps described by Zeil (1993a, b) and use one cylinder as a guiding beacon to the nest. Discussion 1. What determines the bees preferred orientations? These data show that under a variety of conditions bees tend to orient along a roughly north-south axis. Their preferred orientation on return flights mostly matches that assumed on learning flights, as is the case in other insects tested. The resemblance of the bees orientations on learning and return flights is most striking in the distanceorientation plots of Fig. 8B,C, where the return flights seem to replicate in amazing detail the pattern of orientations of the learning flights, a topic that we will consider in more detail elsewhere. In the garden where the horizon is lowest between north and west, bees were mostly oriented northward with some south directed flights. The proportion of south directed flights rose and the distribution became bimodal when the skylight was distributed more evenly by hanging white curtains around the experimental table (Fig. 4C-F). On the roof with one landmark to the north of the nest, bees were mostly oriented south around midday and north in late afternoon and early evening. The data were more variable with four cylinders. South facing predominated around midday and early afternoon, north 13

375 376 377 378 379 38 381 382 383 384 385 386 387 388 389 39 391 392 393 394 395 396 397 398 399 4 41 42 43 44 45 facing increased in frequency during the late afternoon. but in the early evening there was no clear pattern. The effects of time of day and the curtain on orientation suggest that whether bees select the north or the south pole of the north-south axis was set by the distribution of light. By and large, bees chose the brighter pole. A tentative, working hypothesis from these results is that there are two major attractors for orientation north and south - set perhaps by a celestial or a magnetic compass, and that the bees preferred orientation flips between the two attractors according to the distribution of light in the insect s surroundings. Such a mechanism would be one way of implementing a compromise between keeping to a fixed orientation (to allow an efficient use of view-based memories) and keeping the sun broadly within the fronto-lateral visual field (to enhance the detectability of landmarks as suggested by Fig. 1). In summer in the northern hemisphere, the sun rises in the northeast and sets in the northwest so this hypothesis predicts that body orientation on open ground will be to the north on flights early and late in the day. Unfortunately, the early morning weather in May 28 made it impossible to record flights. More experimental work is needed to test the viability of these suggestions. The influence of landmarks close to the nest was in most cases small. The NW cylinder had the clearest effect (Fig. 8). With two cylinders to the west of the nest hole, the bees orientation during learning and return flights was biased away from that set by the postulated compass based attractors. Instead bees faced in the direction of the vector between nest and the NW cylinder. This behaviour is interesting for two reasons. First, it supports the idea that bees store views close to the nest (see also Fig. 2C) by providing the start of an explanation of how the bee sets its body orientation relative to the cylinder. Since the nest-hole is inconspicuous, the heading direction from nest to cylinder is most easily learnt in terms of compass or panoramic cues while the bee stores a frontal view of the cylinder when near the nest. Second, it raises the question of why the bees preferred orientation should be mostly independent of the position of the cylinders (Fig. 7). There was no bias towards a cylinder 14

46 47 48 49 41 411 412 413 414 415 416 417 418 419 42 421 422 423 424 425 426 427 428 429 43 431 432 433 434 435 when bees encountered a complementary array of two cylinders with one to the NNE (Fig. 7E). Nor was it seen with an array of four cylinders, when both the NNE and the NW cylinder were present (Fig.7F). The slight westward bias of orientations with a single cylinder placed just west of north indicatets that this cylinder may also bias body orientation. These results suggest that for a landmark to bias body orientation in its direction several conditions should be met. The cylinder should be close to the stronger of the postulated axial attractors, as is the case for the N (Fig. 7A), NNE (Fig. 7E), and NW (Fig. 8) cylinders. It should be viewed in a direction with a relatively low horizon (as is met by the N and NW cylinders, but not by the NNE cylinder). And its effect should not be suppressed by other significant cylinders near the nest, as seems to happen with four cylinders (Fig. 7F). 2. Preferred orientation and the detection of landmarks We have attempted to tie the pattern of body orientations seen in the learning flights of Bombus terrestris to their trait of nesting on the ground. As suggested by Fig. 1, there is an advantage for insects viewing landmarks against the ground to face towards the sun. Interestingly, there are two reported cases of insects that do not face the sun during learning flights. Vollbehr (1975) finds that honeybees, on their first learning and return flights from and to the hive, do just the opposite and fly with the sun directly behind them. If the bees are kept captive for some hours after their outward flight, they still approach the nest with the sun behind them. Though perverse from the point of view of Fig. 1, this behaviour may be appropriate for the honeybees normal nesting site, which is in tree-trunks well above the ground, where the background to the nest site is likely to be sky or neighbouring tree trunks. The second example is the solitary wasp, Cerceris, which, like Bombus terrestris, is a ground nesting insect. Its orientation is controlled principally by landmarks rather than by compass cues (Zeil, 1993a). Why might these two ground nesting insects differ in this way? Perhaps one reason is that Cerceris catches insects and may have a more prominent zone of high acuity in frontal retina for hunting (Land, 1997). In this case, Cerceris might 15

436 437 438 439 44 441 442 443 444 445 446 447 448 449 45 451 452 453 454 455 456 457 458 459 46 461 462 463 464 gain more from keeping landmarks near to its frontal field than from choosing an orientation that enhances contrast. Thus, as pointed out earlier (Brünnert et al., 1994; Jander, 1979) there are common features, but also marked differences between species. In general, it seems that the design of an insect s learning flights is tuned to accommodate the particularities of its habitat, its behaviour and its sensory systems. In the case of Bombus terrestris, there seem to be preset factors controlling preferred viewing directions that can to some extent be overridden by features of the nest s surroundings. Acknowledgements Financial support came from the EPSRC and BBSRC. O.R. was supported by a de Bourcier doctoral fellowship and a grant from the ORSAS. References Bates, H. W. (1863). The naturalist on the River Amazons. London, John Murray Becker, L. (1958). Untersuchungen über das Heimfindevermögen der Bienen. Z. vergl. Physiol. 41, 1-25. Brünnert, U., Kelber, A. and Zeil, J. (1994). Ground-nesting bees determine the location of their nest relative to a landmark by other than angular size cues. J. Comp. Physiol. A 175, 363-369. Collett, T. S. (1995). Making learning easy: the acquisition of visual information during orientation flights of social wasps. J. Comp. Physiol. A 177, 737-747. Collett, T. S. and Baron, J. (1994). Biological compasses and the coordinate frame of landmark memories in honeybees. Nature 368, 137-14. Collett, T. S. and Lehrer, M. (1993). Looking and learning - a spatial pattern in the orientation flight of the wasp Vespula-Vulgaris. Proc. R. Soc. Lond. B 252,129-134. 16

465 466 467 468 469 47 471 472 473 474 475 476 477 478 479 48 481 482 483 484 485 486 487 488 489 49 491 492 493 494 495 Collett, T. S. and Rees, J.A. (1997). View-based navigation in Hymenoptera: multiple strategies of landmark guidance in the approach to a feeder. J. Comp. Physiol. A 181, 47-58. Jander, R. (1997). Macroevolution of a fixed action pattern for learning: the exploration flights of bees and wasps. In Comparative psychology of invertebrates: The field and laboratory study of insect behavior (ed. G.Greenberg, E.Tobach), pp, 79-99. New York: Garland Land, M.F. (1997). Visual acuity in insects, Annu. Rev. Entomol. 42, 147 177. Lehrer, M. (1991). Bees which turn back and look. Naturwiss. 78, 274-276. Opfinger, E. (1931). Über die Orientierung der Biene an der Futterquelle. Z. vergl. Physiol.15, 431-487. Tinbergen, N. (1932). Über die Orientierung des Bienenwolfes (Philanthus triangulum). Z. vergl. Physiol. 16, 35 334. van Iersel, J. J. A. and van der Assem, J. (1964). Aspects of orientation in the diggerwasp Bembix rostrata. Anim. Behav. Suppl. 1, 145-162. Vollbehr, J. (1975). Zur Orientierung junger Honigbienen bei ihrem 1. Orientierungsflug. Zool. Jb. allg. Zool. Physiol. 79: 33-69. Wagner, W. (197). Psychobiologische Untersuschungen an Hummeln. Zoologica 19, 1-238 Wehner, R. (1981). Spatial vision in arthropods. In Handbook of sensory physiology, VII/6C (ed. H. Autrum), pp. 287-616. Berlin: Springer. Wolf, E. (1926). Über das Heimfindevermögen der Bienen 1. Z. vergl. Physiol. 3, 615-691. Wolf, E. (1927). Über das Heimfindevermögen der Bienen 2. Z. vergl. Physiol. 6, 221-254. Zeil, J. (1993a). Orientation flights of solitary wasps (Cerceris, Sphecidae, Hymenoptera). 1. Description of flight. J. Comp. Physiol. A 172, 189-25. Zeil, J. (1993b). Orientation flights of solitary wasps (Cerceris, Sphecidae, Hymenoptera). 2. Similarities between orientation and return flights and the use of motion parallax. J. Comp. Physiol. A 172, 27-222. 17

496 497 498 499 5 51 52 53 54 55 56 57 58 59 51 511 512 513 514 515 516 517 518 519 52 521 522 523 524 525 Figure legends Figure 1. Landmarks against a horizontal background are most detectable when viewed in the direction of the sun. An upright branch against grass viewed in different directions relative to the sun s azimuth. Top row: camera faces towards the sun and 6 o either side. Bottom row: camera faces away from the sun and 6 o either side. Photographs were taken at about 11:3 in mid-november at a latitude of 5.87 o. Figure 2. Bumblebees facing direction in the garden during learning and return flights with a cylinder placed 8 cm from and 15 o west of north of the nest-hole. A: Frequency distribution of the horizontal orientation of the bee s long axis on all frames of 85 learning flights and of 61 return flights. Bin width is 1 o. Here and in other figs. blue lines (dark) show learning flights and red lines (pale) return flights. The dashed vertical line at o indicates north and the dotted lines at ±18 o indicate south. Distribution wraps around to avoid masking peaks to the south. + and show nest-hole and cylinder respectively with N up. B: Frequency distribution of the peak orientation of each learning and return flight. C and D: Colour map of compass direction against distance from the nest. Data are normalised across each column for learning and return flights. Bin width is 2 cm. C: learning flights, D: return flights. Figure 3. Bumblebees facing direction on the roof during learning and return flights with a cylinder placed 8 cm from and due north of the nest-hole. A: Frequency distribution of the horizontal orientation of the bee s long axis on all frames of 67 learning flights and of 42 return flights. B: Frequency distribution of the peak orientation of each learning and return flight. C to F: Data segregated according to time of day. C: dark blue shows frequency distribution as in A for learning flights recorded before 15:3; light blue for learning flights after 15:3. D: Peak orientations of learning flights in successive 2 hour bins categorised as north, east, south, west (±45 o ). Numbers at top of panel are total flights in each bin. Times below abscissa indicate the starting time of each 18

526 527 528 529 53 531 532 533 534 535 536 537 538 539 54 541 542 543 544 545 546 547 548 549 55 551 552 553 554 555 556 bin. E: Dark red shows frequency distribution of return flights recorded before 16:3; light red for return flights after 16:3. F, as D, but for return flights. Figure 4. Bumblebees facing direction in the garden during learning and return flights with 4 cylinders each placed 2 cm from and N, S, E, or W of the nest-hole. Left column: frequency distributions of body orientation pooled over all frames. Right column: frequency distributions of the peak orientation of each flight. A and B: recordings of 3 learning flights and 27 return flights with no curtain around table. C to F: recordings of 39 learning flights and 43 return flights with curtain. C and D: all frames, E and F: only frames in which bee was less than 7 cm from and faced the nest-hole. Figure 5. Bumblebees facing direction on the roof during 28 learning and 137 return flights with 4 cylinders each placed 2 cm from and N, S, E, or W of the nest-hole. A to F: format as Fig. 3. Figure 6. Body orientation plotted against time of day for learning and return flights recorded in the garden. The peak orientation of each flight in successive one hour bins is categorised as north, east, south or west (±45 o ). Format as in Fig 3D. See text for more detail. Figure7. Body orientation in the garden with different arrangements of cylinders. A-D: One cylinder placed 8 cm in one of four directions from the nest (about 14 o to the west of north, east, south and west). E, F: Two or four cylinders placed 2 cm from the nest. Left panel of each column: distribution of body orientations pooled over all frames of learning and return flights. Right panel: distribution of frames in which the bee faced within 1 o of the nest-hole. Figure 8. Body orientation in the garden with cylinders to the NW and SW of the nesthole. A: Distribution of orientations of all frames, frames in which bees faced the nest, and frames in which they faced the NW or SW cylinder. B, C: Plots of body orientation against distance from the nest when bees faced the NW cylinder or the nest. 19

557 558 559 56 561 562 563 Table 1. Means and vector lengths of the peak orientation of each learning and return flight with nest-hole marked by a single cylinder Table 2. Means and vector lengths of the peak orientation of each learning and return flight with nest-hole marked by two or four cylinders. 2

564 Table 1 Frames included All Nest facing All Nest facing All Nest facing All Nest facing Landmark position North North South South East East West West relative to nest-hole (345 o ) (168 o ) (77 o ) (257 o ) Learning flight mean vector 335 326 5 5 35 357 333 Vector length.53.48.44.46.24.3.27.6 Standard error 8 9 16 16 28 23 31 13 Number of flights 85 79 31 26 34 31 22 21 Return flight mean vector 336 335 357 13 26 8 346 359 Vector length.82.55.77.57.66.25.8.54 Standard error 5 9 8 14 13 4 6 12 Number of flights 61 6 23 23 16 16 34 34 565 566 567 568 569 57 571 572 573 574 575 576 577 578 21

579 Table 2 58 Frames included All Nest facing All Nest facing All Nest facing All Nest facing Facing NW cylinder (292 o ) Landmark N,E,S,W N,E,S,W 4 square 4 square 2 east 2 east 2 west 2 west 2 west arrangement (Fig. 4A) (Fig. 7F) (Fig. 7E) (Fig. 8) Learning flight Mean vector 354 o 357 332 341 333 322 297 285 294 Vector length.81.68.44.74.72.58.61.64.89 Standard error 7 9 12 7 6 8 7 7 3 Number of 3 29 5 45 61 57 66 58 66 flights Return flight Mean vector 355 346 8 357 322 331 297 298 294 Vector length.59.31.56.6.83.78.66.62.86 Standard error 12 25 1 9 5 5 7 8 4 Number of flights 27 27 4 4 56 56 52 52 52 581 22

Fig 1

+ A.8 B.6.4.2.1.5-18 -9 9 18-18 -9 9 18 C Learning flights O rientation (degrees) Orientation (degrees ) D 18 9 9 1 2 3 4 5 6 Distance (cm) Return flights 18 9 9 1 2 3 4 5 6 Distance (cm).1.1 Fig. 2

+ A.4.3.2.1 B.8.6.4.2 F requenc y C.4.2-18 -9 9 18.4.2-18 -9 9 18 Orientation (degrees) Learning flights D.6 38 5 24 E -18-9 9 18 Return flights F N E SW N E SW N E SW 14 16 18.6.6 13 1 19.4.4.2.2-18 -9 9 18 Orientation (degrees) N E SW N E SW N E SW 14 16 18 Time (hours) Fig. 3

+ A.6.4.2 B.2.15.1.5 F requenc y C.4.3.2.1 E.8.6.4.2-18 -9 9 18-18 -9 9 18 D.1.8.6.4.2 F.1.5-18 -9 9 18-18 -9 9 18-18 -9 9 18-18 -9 9 18 Orientation (degrees) Fig. 4

+ A B.4.3.2.1.8.6.4.2 C -18-9 9 18 Learning flights D -18-9 9 18 Orientation (degrees) 9 78 48 53 27 F requenc y.3.2.1.4.3.2.1 E.6-18 -9 9 18 Return flights F.6 NESW NESW NESW NESW NESW 11 13 15 17 19 33 12 45 23 23.4.4.2.2-18 -9 9 18 Orientation (degrees) NESW NESW NESW NESW NESW 12 14 16 18 2 Time (hours) Fig. 5

A n= 6 35 23 36 44 42 4 12 26 12.8.6.4.2 Frequency B.8 NESW NESW NESW NESW NESW NESW NESW NESW NESW NESW 1 11 12 13 14 15 16 17 18 19 n= 14 22 47 43 46 46 12 13 11 5.6.4.2 NESW NESW NESW NESW NESW NESW NESW NESW NESW NESW 11 12 13 14 15 16 17 18 19 2 Time (hours) Fig 6.

+ + + + + + A.8.6.4.2 All frames Nest facing All frames Nest facing B.8.6.4.2-18 -9 9 18.5.4.3.2.1-18 -9 9 18-18 -9 9 18-18 -9 9 18.6.4.2 F requenc y C.6.4.2-18 -9 9 18.8.6.4.2-18 -9 9 18 D.1.8.6.4.2-18 -9 9 18.8.6.4.2-18 -9 9 18 E.6.4.2.1.6.8.4.6.4.2.2 F.8.6.4.2-18 -9 9 18-18 -9 9 18-18 -9 9 18-18 -9 9 18 Orientation (degrees) Fig. 7

+ A.5.4.3.2.1 All frames.6.4.2 Facing nest Facing NW cylinder -18-9 9 18-18 -9 9 18-18 -9 9 18-18 -9 9 18.2.15.1.5.25.2.15.1.5 Facing SW cylinder B Orientation (degrees) 18 9-9 Learning flights when facing NW cylinder Orientation (degrees) 18 9 9 Return flights when facing NW cylinder.2 1 2 3 4 Distance from nest (cm) 1 2 3 4 Distance from nest (cm) C Orientation (degrees) 18 9 9 Learning flights when facing nest Orientation (degrees) 18 9 9 Return flights when facing nest.125 1 2 3 4 Distance from nest (cm) 1 2 3 4 Distance from nest (cm) Fig. 8