Flies land upside down on a ceiling using rapid visually mediated rotational maneuvers

Behavioral modules in successful inverted landing

Fruit flies (Drosophila) land on vertical surfaces by continuous deceleration with negligible body rotation before the touchdown (8, 14). In contrast, blue bottle flies (Calliphora) landing on a ceiling exhibited a sequence of four behavioral modules—started with an upward acceleration, followed by a rapid body rotational maneuver and leg extension, and ended with a leg-assisted body swing with forelegs firmly planted on the ceiling, thereby orienting the fly’s body ventral side up (i.e., inverted) (Fig. 1 and fig. S3, Aii and Bii). The process from the start of the body rotation to the ventral side up landing lasted approximately four to eight wingbeats in all the successful landings (n = 18; table S1) (average wingbeat frequency, 172.7 ± 7.7 Hz).

In successful inverted landings, we observed substantial kinematic variations in the axes of rotation and the magnitude of rotational maneuvers, as well as in the degree of leg-assisted body swing. Thus, flies used a wide range of maneuvers when landing on a ceiling, which we categorized into pitch dominated, roll dominated, pitch and roll combined, and longitudinal or lateral body swing dominated (movie S1 to S5 and table S1). Examples of three typical sequences are shown in Fig. 1 (D to F), illustrating parts of this variation. In the first two examples, flies used rapid rotational maneuvers primarily about pitch (Fig. 1D and movie S1) or roll (Fig. 1E and movie S2), which oriented their body to a nearly inverted orientation before touchdown. Leg extension occurred slightly after the start of the rotational maneuvers (horizontal black dashed lines, Fig. 1, Di and Ei), and the body swinging phase followed after legs were planted on the substrate. Notably, the average peak angular rate of the rotational maneuvers reached approximately 4000°/s (fig. S3, Av and Bv) and could be as high as 6000°/s (Fig. 1Eiii), greater than those reported in the escape maneuvers of fruit flies (1) and hummingbirds (15). In the third example, flies relied almost entirely on the leg-assisted body swing to reorient and landed with negligible rotational maneuver (Fig. 1F and movie S3), similar to previously reported landing on vertical surfaces (4, 8). Thus, compared with the first two, this landing strategy likely leveraged more of the mechanical and structural processes (e.g., adhesion due to pulvilli or damping due to viscoelasticity of leg) (16), which assisted the transfer of the body’s linear momentum to rotational momentum.

Kinematic differences between successful and failed landings

We observed both successful and failed landings. A subset of flies landed in a smooth and coordinated fashion (successful landings), whereas others failed to land properly and collided head-on upon approach (failed landings). In successful landings, the four behavioral modules collectively led to a proper combination of body inversion and linear velocity with properly positioned legs prior to touchdown.

To quantify how well a fly’s body was oriented, we calculated the degree of inversion (DoI), which measures the degree to which the fly’s body is aligned with respect to a fully inverted orientation before touchdown (see Materials and Methods). DoI ranges from 0, representing no body inversion (or ventral-side down), to 1, representing full body inversion (or ventral-side up). In successful landings, the DoI increased with vertical velocity (Fig. 2A) but decreased with the forward velocity (Fig. 2B). This indicated that flies needed to be more inverted when their upward velocity was greater or when their horizontal velocity was lower. In most failed landings (total N = 15 analyzed; table S1), the flies were insufficiently inverted before touchdown, as compared with successful landings (Fig. 2). The failure in these landings can be attributed to delayed or minor body rotation prior to touchdown (Supplementary Materials). Consequently, these flies could not properly land on the ceiling but instead collided head-on. Notably, flies also failed at landing due to early rotation (see movie 7), as the early inversion led to insufficient vertical velocity to reach the ceiling (“+” in Fig. 2). Last, in some failed landings, flies had similar inversion to that of successful landings, but failed regardless. This happened due to delayed leg extension, which resulted in an improperly positioned leg to have the tarsi firmly planted on the substrate (see movie 8).

Yet, even after failed attempts, flies could still recover and land, using groping landing (n = 24; table S1). In this type of inverted landing, flies hover or fly at low vertical speed underneath the ceiling (Fig. 2), then they grope for the ceiling using their forelegs, and with a firm grip on the substrate, swing their body up to land (movie S6).

The kinematic complexities and variations in inverted landings indicate that they may involve more diverse neural processes than those reported for triggering leg extension and deceleration (8, 12, 14). In particular, the rotational maneuver with variable pitch and roll for a proper inversion was the major determining factor for success. Thus, in addition to being triggered, it is likely that the rotational maneuvers were mediated by sensory cues in a short period immediately before touchdown. We next examined the potential sensory cues and their roles in triggering and mediating the rotational maneuvers, based on the statistical analyses of the kinematic data.

Triggering of rotational maneuvers

We first describe the putative visual cues that flies could sense as they approach the ceiling with three components of linear velocity (vertical V_z, fore/aft V_x, and lateral V_y; Fig. 1C). Note that our study does not consider feature-related triggering (e.g., object retinal location) (8), because, ideally, the use of uniform mesh grids should generate coherent motion over the entire retina when the fly is sufficiently close to the ceiling. Previous work on insect visual control (8, 14, 17) emphasized the importance of three types of visual cues. First is the relative retinal expansion velocity (RREV) due to looming stimuli (Fig. 3), which can be calculated as the ratio of a target’s expansion rate to its size on the fly retina during upward translation (V_z). RREV also corresponds to the reciprocal of a physical variable called time to collision τ, which represents the time to collision, assuming constant velocity (14). RREV is considered an important perceptual cue that controls the approaching velocity during landing or obstacle avoidance (18, 19). Second is the relative fore/aft angular velocity (or translational optic flow) of the ceiling on the fly retina (ω_y) (Fig. 4Ai). These visual cues result from the body fore/aft translation (V_x) and were found previously to control the grazing landing on horizontal surfaces (17). Third is the relative lateral angular velocity (or translational optic flow) of the ceiling ω_x on the fly retina (Fig. 4Bi), resulting from lateral translation (V_y). We calculated the time traces of these three visual cues in both the yaw-aligned global frame and the retina-fixed frame (fig. S4) using kinematic variables according to their mathematical relationships (fig. S1). All three visual cues could be calculated directly from the optic flow [see Supplementary Materials and (19)].

We assumed that the rotational maneuvers were triggered after a fixed time delay when the triggering visual cue reached a threshold (14). This is arguably the most parsimonious predictive model for the triggering and enabled us to test whether the above visual cues are related to the onset of the rotational maneuvers as described below.

We calculated the time traces of the coefficient of variance (CV) for each visual cue using trials in which flies landed successfully in their first attempt on a high-contrast landing area (table S1). The results for the RREV are shown in Fig. 3, while the complete results are shown in figs. S4 and S5 (Supplementary Materials). The CV measures the dispersion of a variable defined by the ratio of its SD and mean. The triggering of the pitch and roll motion was analyzed separately by aligning data from different landing trials at either the start of the pitch or roll. If the rotational maneuvers were triggered after some time delay when a visual cue reached a threshold value, the CV of this visual cue should be at its minimum. In addition, it should be lower than those of other nontriggering visual cues (14), i.e., the triggering visual cue should have the least dispersion (see Materials and Methods). For example, in a previous study on the triggering of linear deceleration in houseflies (Musca domestica L.) landing on vertical posts (14), the CV of the RREV is approximately 30%, lower than the other variables. Here, among the three visual cues tested, the RREV in the yaw-aligned global frame had the least CV for triggering either pitch (15%) or roll (25%) rotations (Fig. 3B), whereas the CV of the other two visual cues were significantly larger (figs. S4, A to C, and S5, A to C). This indicated that RREV was the primary visual cue triggering rotational maneuvers in inverted landing, similar to the triggering of deceleration for landing on vertical posts. Note that the CV of distance between the fly and the ceiling was close to that of the RREV (figs. S4D and S5D); however, flies are thought to not be able to directly measure distance (20, 21).

The CV of the RREV was minimum, approximately between 7 ms (ΔT_l) and 27 ms (ΔT_u) before the onset of rotational maneuvers (Fig. 3B), suggesting that the visual latency ΔT between the instant of perception and the onset of a maneuver was within this small range. Although the latency inferred from this analysis was smaller than estimated from other behavioral studies, e.g., in linear speed control (22) and escape maneuvers (1), it fell within the range of latency in a fly’s visuomotor pathway (23, 24). Last, the corresponding RREV threshold value (Fig. 3B) indicated that the critical time to collision, below which the rotational maneuver was likely to be triggered, was between 31 and 53 ms. This threshold is lower than the one previously identified for triggering the prelanding deceleration in houseflies landing on vertical posts, which was approximately 76 ms (14).

Sensory mediation of rotational maneuvering patterns

Rotational maneuvers were highly variable in terms of their roll and pitch rates, which in successful landing have led to a proper body inversion according to the flies’ body linear velocity (Fig. 2). This suggested that, in addition to the onset timings, the patterns of the rotational maneuvers were also mediated by sensory cues. Flies are known to use sensory cues to mediate rotational maneuvers via both feedforward and feedback pathways (25). The feedforward path issues spontaneous steering commands that produce large, transient wing motion changes, which determine the initial response magnitude of the maneuver (25, 26). The feedback path mediates compensatory reaction (e.g., haltere-mediated feedback) that produces fine-scaled wing motion change, which determines the active damping and stabilizes the maneuver (2, 25, 26). Here, we identified the potential sensory cues and their roles in the feedforward mediation of the rotational maneuvers. This was achieved by identifying the correlations between the peak roll and pitch rates of the rotational maneuvers with the sensory cues perceived by flies prior to or slightly after the onset of the maneuvers. Note that these sensory cues were mainly dependent on the flies’ body linear velocities shortly before the onset of rotational maneuvers, when body angular velocity was negligible.

Specifically, we evaluated correlations between the peak pitch and roll rates with a set of putative sensory cues at varying time instants prior to or slightly after the onset of rotational maneuvers (Fig. 4, and more details in figs. S6 and S7 and table S2). The correlations using the sensory cues 20 ms before the peak angular rates are shown in fig. S8 as an example. We used angular rates rather than angular acceleration because previous studies suggested that motor responses (i.e., wing kinematic control variables) were more strongly correlated with body angular rates due to the periodic and highly damped nature of flapping flight (27, 28). The putative sensory cues included the three visual cues tested above for the triggering (RREV, ω_x, and ω_y) and the three components of body linear velocity (V_x, V_y, and V_z), which were likely perceived via mechanosensory modalities of flies (29). Linear regression analysis showed that, within a large time period prior to the onset of the maneuvers (P < 0.05, Student’s t test), the peak pitch rate was positively correlated with RREV (Fig. 4Aii) and vertical velocity V_z (Fig. 4Aiii), but negatively correlated with fore/aft ceiling rotation ω_y (Fig. 4Aii) and fore-aft linear velocity V_x (Fig. 4Aiii). Similarly, peak roll rates were positively correlated with the lateral ceiling rotation ω_x (Fig. 4Bii) and lateral linear velocity V_y (Fig. 4Biii), also within a large period (P < 0.05, Student’s t test) prior to or shortly after the onset of the maneuvers. The above correlation analysis supports the possibility of sensory mediation of rotational maneuver patterns, with pitch mediated by sensory cues encoding upward and fore/aft translation, and roll mediated by sensory cues encoding lateral translation.

The above correlation analysis also raised the possibility that flies modulate the magnitude of rotational maneuvers to exploit leg-assisted body swing, including transfer of linear to angular momentum. Specifically, flies exhibited a reduced pitch rate when forward linear momentum was high, as indicated by the negative correlation between peak pitch rate with sensory cues resulting from forward translation (visual ω_y or mechanosensory V_x). Thus, instead of active pitching, flies inverted their body by increasing the leg-assisted longitudinal body swing and the transfer of forward linear momentum to pitch angular momentum (Fig. 1F). On the other hand, flies exhibited an increased roll rate when the lateral linear momentum was high, as indicated by the positive correlation between peak roll rate and sensory cues resulting from lateral translation (visual ω_x or mechanosensory V_y). This opposite correlation may result if flies need sufficiently large banking to raise their ipsilateral legs high enough to reach the ceiling for the leg-assisted body swings (middle legs cannot extend upward as high as forelegs without banking the fly’s body). Thus, this strategy could also assist the execution of lateral body swings that transferred lateral linear momentum to roll angular momentum.

Wing kinematic patterns that produce pitch and roll

The execution of the rotational maneuvers requires sensorimotor transduction of sensory cues to motor outputs that help modulate wing motion to generate various aerodynamic forces and moments. We next examined the changes of wing motion responsible for generating pitch and roll during rotational maneuvers. The wing motion patterns underwent moderate changes that lie between those reported in fruit flies (1) and hummingbirds (27) during escape maneuvers. We observed the following changes. First are the bilaterally symmetric changes in wing spanwise rotation (Fig. 5, A and Ci) and bilaterally symmetric changes in stroke plane angle (Fig. 5, A and Cii), which were both strongly correlated with the pitch rate (for example, to produce nose-up pitch, the fly tilted its stroke plane and shifted the mean rotation angle backward). Second are the bilateral differences in wing deviation (Fig. 5, B and Ciii), which were strongly correlated with the roll rate. The bilateral differences in wing deviation, which resulted in the lateral tilt of stroke plane, have also been observed in escape maneuvers in hummingbirds (27) and fruit flies (1). In addition, bilateral differences in wing stroke amplitude and mean wing rotation were observed and likely contributed to the roll. More details of the variables of wing motion changes and their correlations with body angular rates are provided in table S3. Despite the size and physiological differences, the variables of wing motion changes for generating pitch and roll during the inverted landing in flies closely resemble those of hummingbirds during escape maneuvers (27).