Role of outstretched forelegs of flying beetles revealed and demonstrated by remote leg stimulation in free flight

The flight posture of many insects, including butterflies, locusts, dragonflies, moths and bees, is commonly described as the legs being folded or pressed closely against the body during flight (Borst, 1986; Burrows, 1996; Nachtigall, 1974). In contrast, beetles fully lift and outstretch their forelegs while inflight (Fig. 1). Moreover, many beetle species have thick, long forelegs, unlike most flying insects (Van Truong et al., 2014). Naively, flying with outstretched legs would seem inadvisable, as it would tend to increase drag (Combes and Dudley, 2009). Meanwhile, beetles often swing their forelegs while inflight. According to the physical principle of conservation of angular momentum, when a leg rotates (swings) about the leg base (coxa), the body of the flying beetle should rotate in the opposite direction (Mountcastle et al., 2015). A long and relatively heavy foreleg will have a relatively large moment of inertia so that the resulting torque exerted on the body might be large enough to significantly rotate the body inflight.

As reported in many insect species, body structures apart from the wings could also contribute to the flight control, and function as auxiliary maneuvers (Wagner, 1986; Taylor and Thomas, 2003; Camhi, 1970; Rowell, 1988; Zanker et al., 1991). Specifically, it is reported that both the abdomen and the hindlegs of the locust are effective in assisting with flight turnings and compensating for flight fluctuations by swaying them laterally (Camhi, 1970; Rowell, 1988; Arbas, 1986). Similarly, Zanker (1988) demonstrated by visual stimulation that the abdomen of flies takes an active role in flight directional control. The honeybee widely manipulates its body streamline to adjust the flying speeds and heading directions (Luu et al., 2011; Nachtigall, 1974). Meanwhile, the bees make use of their hindlegs to stabilize their body in flight (Combes and Dudley, 2009; Mountcastle et al., 2015). It has also been found that the elytra of beetles are placed asymmetrically in flight turnings to produce uneven lift (Van Truong et al., 2012). Even the rotation of the head has been found to visually coordinate the flight turnings in locusts (Hensler and Robert, 1990; Miall, 1990). We believe the stable and efficient flight of insects cannot be separated from the diverse auxiliary maneuvers.

Here we show that the forelegs are ‘intentionally’ outstretched and swung to facilitate turning while inflight. The beetle turns its body (the direction of propulsion) by swinging its forelegs. Leg motion tracking and electromyography (EMG) in tethered flight showed that the forelegs were voluntarily swung clockwise in yaw to induce counter-clockwise rotation of the beetle body for left turning, and vice versa. The yaw torque generated by leg swing was measured, which was proved large enough to rotate the body. Furthermore, we demonstrated remote control of left–right turnings in flight by swinging the forelegs via a remote electrical stimulator. As the wings are the dominant mechanism for controlling insect flight (Chapman et al., 2012), the present study indicates that the outstretched foreleg plays a supplemental role in steering during flight.

The animals used in this study were adult male Mecynorrhina torquata (Drury 1782) beetles (order Coleoptera; length: 62±8 mm; mass: 7.4±1.3 g) that were fed with beetle jellies twice a week. The rearing room was maintained at a temperature and humidity of approximately 25°C and 60%, respectively. Beetles that could fly freely for more than 10 s were selected for the experiments. The use of this animal was permitted by the Agri-Food and Veterinary Authority of Singapore (AVA; HS code: 01069000, product code: ALV002). Invertebrates, including beetles, are exempt from ethics approval for animal experiments according to the National Advisory Committee for Laboratory Animal Research (NACLAR) guidelines.

Each beetle was anesthetized in a small sealed bag containing CO₂ for 1 min. Next, the legs were constrained by a rubber band. Two tiny holes were pierced through the cuticle above the target muscle using insect pins (enamel-coated #5, Indigo Instruments, Waterloo, ON, Canada). The electrodes consisted of 10-cm segmented Teflon-insulated silver wires (127 μm uncoated diameter, 178 μm coated diameter; A-M Systems, Sequim, WA, USA). One side of the silver wire was flamed to remove the insulated layer and expose the silver layer. The flamed ends were inserted through the tiny holes to a depth of approximately 3 mm. Melted beeswax was used to cover the holes because beeswax quickly solidifies and thus immobilizes the silver wires.

As demonstrated in previous studies, visual stimulation (optical flow of dark and bright stripes) can induce fictive turnings in flying insects (Götz and Wenking, 1973; Luu et al., 2011; Zanker, 1988); thus, we chose this type of visual stimulation to determine the steering ability of beetle forelegs. The beetle was tethered to constrain its flight within the range of a universal coupler (Fig. S1). Thus, the beetle was capable of rolling or pitching its body, but yaw rotation was restricted. The beetle was placed ∼20 cm in front of a translucent screen, which was used for projecting the wide-field optical flow patterns (dark and bright stripes) that moved leftward or rightward (Sato et al., 2015). The stripes are 35 mm in width with a contrast rate of 2.5 Hz. In left stimulations, the stripes were moving from right to left and induced leftward turnings, and vice versa. Both left and right visual stimulations lasted 10 s, and the presentation of the two stimulations was alternated. There was a 5 s interval between the stimulations. Thus, one complete visual cycle lasted 30 s.

To track the locomotion of both forelegs, three retro-reflective markers were placed on the beetle, and a referential marker was placed at the rotation center of the coupler. As shown in Fig. S1, one marker was placed at the posterior end of the pronotum and the other two markers were placed at the tips of both foreleg tibias. Moreover, the distances between the foreleg coxae and pronotum marker were measured. A motion capture system (Vicon, Oxford, UK) consisting of six T40s cameras with a resolution of 4 megapixels (2336×1728 pixels) was used to detect the 3D coordinates of the markers by tracking the retro-reflective markers (Manecy et al., 2015; Thies et al., 2007). The coordinates exported from Vicon were recorded at 150 Hz and synchronized with the visual stimulation.

EMG measurement was conducted under visual stimulation. Two silver wires were implanted into the tissue of the target muscle to collect muscular potentials (unilateral EMG) during tethered flight (Sato et al., 2015). To avoid collision with the wings, the wires were spread along the coupler. The two non-implanted ends of silver wires were connected to the input of a signal amplifier (LT1920, Linear Technology Corporation, Milpitas, CA, USA) using alligator clips. The amplified signal was transmitted to a microcontroller-based (CC2430, 7×7 mm², 130 mg, 32 MHz clock, 2.4 GHz IEEE802.15.4-compliant RF transceiver, Texas Instruments, Dallas, TX, USA) development board (SOC_BB 1.1 and CC2430EM 1.2, Chipcon AS, Oslo, Norway). Using the A/D converter on the microcontroller, the collected data were digitalized for wireless transmission based on the IEEE 802.15.4 protocol. The EMG signal of the left protraction muscle was measured and analyzed (Fig. 3A). To avoid possible interference, the right protraction muscle was also implanted with silver wires. The recorded electrical signals were processed by a custom program. The EMG spikes were selected based on 5-sigma control limits and synchronized with visual stimulations. Specifically, the EMG spikes that occurred during the left and right visual stimulations and intervals without stimulation were assigned to their corresponding groups to compare the occurrence rate.

Torque measurements of the body were collected with a torque sensor (Nano17 Titanium, ATI Industrial Automation, Apex, NC, USA) in tethered condition (Taylor and Thomas, 2003). The sensor was fixed vertically downward to the ground, and a custom holder was connected right below the sensor. The holder was designed to suspend the beetle at its pronotum. The z-axis of the coordinate system was vertically upward while the x-axis and y-axis were pointing forward and leftward, respectively. As measured with the inertia sensor and the method proposed by Li et al. (2016), the pitch angle of beetle body in flight was 28.38±3.65 deg (N=5 beetles, n=25 flights; all data are represented as means±s.d.). Thus, we adjusted the pitch angle of tethered beetle to 28 deg. The beetle was stuck to the holder by beeswax and two thin silver wires were implanted into the left foreleg protraction muscle. The two non-implanted ends of silver wires were connected to the output port of a function generator (33220A, Agilent Technologies, Santa Clara, CA, USA) using alligator clips. Prior to stimulation, the left foreleg was forcibly spread out to its flight position.

The free-flight experiment was completed in a motion capture laboratory (dimension: 16×8×4 m). The laboratory was equipped with a motion capture system (Vicon) containing twenty T40 s and T160 cameras fixed along the upper edge of the room (Fig. S3B). The custom-made computer software (BeetleBrain v.0.99b) was used to generate signal commands via a guidance and inertial navigation assistant (GINA, provided by Professor Kris Pister's laboratory at the University of California, Berkeley) base station (2.4 GHz IEEE 802.15.4 wireless protocol). The commands were received and processed by an electrical wireless backpack described by Sato et al. (2015). Electrical signals were generated using two independent channels into the left and right protraction muscles. Each muscle was implanted with a pair of silver electrodes including a working electrode and a counter electrode. The muscles were unilaterally stimulated in the experiment. A rechargeable lithium ion battery (3.7 V, 350 mg, 8.5 mAh; Micro Avionics, Horsely, Derbyshire, UK) was connected to the backpack, and the surface was wrapped with retro-reflective tape (Silver-White, Reflexite Corporation, Oranienburg, Germany). The backpack assembly was attached to the pronotum of the beetle to enable the detection of 3D flight trajectories using the motion capture system (Fig. S3A).

In the free-flying experiments, the backpack assembly was used to stimulate both foreleg protraction muscles. The electrical stimulation of the left or right protraction muscle induced the swing of the corresponding leg clockwise or counterclockwise (viewed from the dorsal side of the beetle). A preliminary experiment was conducted to determine the appropriate stimulation voltage for the protraction muscle. The function generator was used as the signal source, which generated pulse signals with a 100 Hz frequency, 10% duty cycle and 500 ms duration. The voltages ranged from 0.5 to 1.5 V with a step width of 0.1 V. While stimulating the foreleg protraction muscles, the EMG of flight muscles was recorded. Five flight muscles were measured, including the dorso-longitudinal muscle, dorso-ventral muscle, basalar muscle, subalar muscle and second axillary muscle. The minimum voltage that elicited regular EMG spikes on any flight muscle was defined as the threshold. The overall threshold voltage was 0.90±0.07 V (N=5 beetles, n=5 thresholds). Accordingly, the amplitude of all electrical stimulations was set to 0.7 V.

Once a beetle starts to fly, whether in free or tethered flight, its forelegs extend (Fig. 1). We hypothesized that the unique posture of a beetle may have some effects on its flight control, especially on the turning control. Thus, we conducted leftward and rightward visual stimulation of tethered beetles using optical flow of dark and bright stripes to induce fictive turns (Götz and Wenking, 1973). To study the correlation between turning and foreleg motion, we tracked foreleg motion during visual stimulation using a 3D motion capture system (Vicon; Fig. S1). During the fictive turns, the horizontal swinging of extended forelegs was frequently observed (Fig. 2A). Within a sample size (N=10 beetles, n=80 left fictive turns, n=80 right fictive turns), we found that the forelegs mostly swung clockwise (from the view of the beetle's dorsal side) to produce fictive left turns and counterclockwise to produce fictive right turns (P<0.0001, binomial test; Fig. 2B).

We speculated that the leg swings associated with fictive turns were actively induced by the beetle itself, i.e. visually stimulated beetles exhibit optomotor responses, including the activation of leg muscles, to produce swinging. To clarify whether the leg swings were produced actively or passively, we used EMG to assess the protraction muscle of the left leg (Fig. 3A) during visual stimulation. The contractions of the muscle in the left foreleg cause it to swing clockwise (Cao et al., 2014). Indeed, a one-sample t-test indicated the majority of EMG spikes in the muscle occurred during left fictive turns (N=5 beetles, n=5 EMG recordings, t₄=10.82, P=0.0002; Fig. 3B,C). This result suggests that the foreleg swings during fictive turns (Fig. 2) are not induced by external forces but rather by the tension created by leg muscle contraction.

The yaw torque exerted on a beetle's body during a leg swing effectively rotates the body. We tethered a beetle on a torque meter and electrically stimulated the protraction muscle of the left foreleg to produce clockwise swinging (N=4 beetles, n=40 trials). It is known that the pitch angle mainly correlates with the longitudinal flying speed and does not apparently influence on the horizontal turning on flapping wing flyers (Cheng et al., 2011, 2016). Thus, we focused on the analysis of yaw torque and roll torque. The results revealed that the induced torque in yaw was as much as ∼7 μN m whereas the induced torque in roll was relatively small, ∼2 μN m (Fig. 4Ai,Bi). Meanwhile, the moment of inertia estimated from the beetle model is 12.36 g cm² in yaw and 7.73 g cm² in roll. Thus, the induced angular displacement was calculated as 1.62 deg in yaw and 0.69 deg in roll within 200 ms. We repeated the same experiment and analysis after removing the legs from the body. The measured torque was smaller, indicating that the significant torque observed prior to leg removal was solely due to foreleg swinging (N=4 beetles, n=40 trials; Fig. 4Aii,Bii).

As further support of the role of forelegs inflight, we demonstrated that exogenous stimulation of the leg muscles induces foreleg swinging and subsequent turning while in free flight (Fig. S3). A radio-controlled backpack (Fig. S3) was mounted on the beetle, and the protraction muscles of the left and right forelegs were alternatively stimulated to induce the clockwise swing of the left foreleg and the counterclockwise swing of the right foreleg, respectively. As expected during free flight, the beetle turned left when the left foreleg was stimulated to swing clockwise, and vice versa (N=5 beetles, n=162 left stimulations, n=184 right stimulations, P<0.0001, binomial test; Fig. 5B,Ci,Di). Because of the electrical stimulation of left or right foreleg protraction muscle, the mean induced turning rates were 3.29±12.71 and −5.42±11.57 deg s⁻¹, respectively (the positive or negative value indicates that the turning rate for left or right turns increases). We confirmed that the electrical stimulation of the protraction muscles solely induced leg swinging and did not affect flight muscles (Fig. S4). Interestingly, once the forelegs were removed from the beetle, left and right turns occurred at a similar rate regardless of which side was stimulated (N=5 beetles, n=90 left stimulations, n=90 right stimulations; Fig. 5Cii,Dii), with mean rates of −0.38±12.09 and −2.35±9.60 deg s⁻¹, respectively. According to a t-test, the difference in turning rates between intact and amputated beetles is considered statistically significant under both left (t₂₅₀=2.23, P=0.013) and right (t₂₇₂=2.18, P=0.015) electrical stimulations. Thus, the left–right turns shown in Fig. 5Ci,Di were caused by the swinging of the left or right foreleg.

In flight, many insects fold their forelegs tightly close to the body, which naturally decreases drag or air resistance, whereas flying beetles stretch out their forelegs for an unknown reason. We hypothesized that the forelegs are ‘intentionally’ outstretched and swung to facilitate turning while inflight and that the beetle turns its body orientation (the direction of propulsion) by swinging its forelegs. To test this hypothesis, we used kinematic and physiological analyses to determine whether swinging the legs during flight was regulated, and we measured the torque exerted on the body during leg swinging using tethered beetles. Furthermore, we induced left and right turns in freely flying beetles by electrically stimulating their leg muscles to produce a swinging motion. This stimulation was achieved by mounting our custom-designed miniature wireless communication device (remote stimulator backpack) on flying beetles.

Through visually inducing fictive turns on tethered beetles, we revealed that the forelegs showed certain swinging motions in accord with the directions of the visual stimulations. Specifically, the beetle voluntarily swings the forelegs clockwise or counterclockwise while inflight to turn in the opposite direction. By monitoring the EMG signals from the left foreleg protraction muscle, whose activation generates a clockwise swing of the left foreleg, we found that the spikes appeared much more frequently in the left visual stimulations than in the right stimulations, which gives the evidence that the swinging motions of forelegs were actively induced by the tension created by muscle contraction rather than passively from some external forces. Thus, beetles voluntarily manipulate their forelegs to produce fictive turns in response to visual stimulation.

Meanwhile, the torque measurement showed that leg swing induced yaw torque on a beetle's body, which would lead to an angular displacement in yaw of up to 1.62 deg within 200 ms. This finding implies that the torque generated by leg motion is significant enough to rotate the heading direction of the beetle. Because the left foreleg was stimulated to swing clockwise in the experiment, the body tended to rotate counterclockwise as a consequence. The rotating direction of the body is in accord with the observations in visual stimulation. As a comparison, the induced roll torque would generate a 0.69 deg roll displacement within 200 ms. As a linear relationship between roll angle and yaw angular velocity was found in beetles (Li et al., 2016), the roll displacement (0.69 deg) would correspond to a −2.29 deg s⁻¹ angular velocity in yaw, which is smaller than the effect of yaw torque (∼13 deg s⁻¹; Fig. 4A). Moreover, the yaw angular velocity induced by clockwise leg swing rotated the body leftwards, which was in accord with the direction of visual stimulation when the forelegs were swinging clockwise. Thus, the leg swing in the turnings should be employed for the yaw turn rather than the bank turn. The use of body posture to change the flying direction by exerting additional torque on the body was previously reported in insects (Berthé and Lehmann, 2015; Zanker et al., 1991). Thus, we believe the beetle swings the forelegs clockwise or counterclockwise in order to generate a yaw torque to rotate its heading direction towards the fictive turns.

The aforementioned findings from the tethered experiments verified our hypothesis that forelegs are swung to facilitate turning. As further support of our hypothesis, we demonstrated that exogenous stimulation of the leg muscles induces foreleg swinging and subsequent turning while in free flight (Fig. S3). In accord with the results of the tethered experiments, the swinging of the left leg (clockwise) induced a leftward turning and the swinging of the right leg (counterclockwise) induced a rightward turning. After leg amputation, the leftward and rightward turnings showed negligible differences in occurrence rate. The electrical stimulations were exactly applied on the leg protraction muscles without influencing any flight muscles (Fig. S4). These results indicate that the induced turnings are solely because of the swinging motion of forelegs. Moreover, the induced turning rates calculated from the free flights match well with the simulation result based on the yaw torque (3.84 deg s⁻¹), which further verifies that the turnings are induced by the induced yaw torque. As known from visual stimulation that forelegs are voluntarily swung in turnings, we demonstrate that beetles manipulate their forelegs as a mechanism of flight directional control. However, the induced turning rates are relatively small. We understand that the range of leg swing is constrained by the structural limit of the leg coxa and that the leg motion cannot sustain a sharp or long-lasting flight turning. This implies that the forelegs may not be dominant when the beetle is performing sharp turnings. Compared with the wings, the leg motion reveals its advantages in response time and precision. Thus, we believe that the legs serve as a supplementary mechanism to generate small directional corrections or initiate a turning in flight.

It is undeniable that wings, as well as their articulations and flight muscles, are the dominant mechanism for controlling insect flight (Dickinson, 2006; Chapman et al., 2012; Sato et al., 2009, 2015). The operating principles of wings have been well studied in various insects (Zanker et al., 1991; Wang et al., 2003). In fact, beetles flew well even after their legs were removed (Fig. 5Dii). However, the present study indicates that the outstretched foreleg plays a supplemental role in steering during flight. Auxiliary flight control by body parts other than wings has been established in some insects (Baader, 1988; Dyhr et al., 2013; Zanker, 1988; Luu et al., 2011; Camhi, 1970; Combes and Dudley, 2009; Lorez, 1995; May and Hoy, 1990; Hensler and Robert, 1990; Berthé and Lehmann, 2015). For example, flies, locusts and honeybees can sway or twist their abdomen to facilitate turning during flight or change their body posture to adjust their flight speed (Baader, 1988; Dyhr et al., 2013; Zanker, 1988; Luu et al., 2011; Camhi, 1970). The abdomen of a beetle is relatively rigid and short; thus, it maybe not feasible or effective to manipulate the abdomen as a method of steering control during flight (Crowson, 2013; Lawrence and Newton, 1982). The forelegs of beetles are relatively large, wide and thick when compared with those of other insects (Fig. 1); however, this design may not be primarily for steering while flying but rather for direct use, such as dirt digging. If these large legs were folded closely to the body, they would be useless in flight. Instead, the beetles outstretch and swing their forelegs to facilitate turning during flight. In this study, we assessed only the forelegs; however, the middle and hind legs may have a similar function because they are also outstretched while the beetle is flying (Fig. 1). Collectively, future studies on the effect of wings and other non-wing body parts on flight should clarify insect flight mechanisms and provide novel insights for the design of insect-scale robotic flapping flyers.

The authors thank Mr Roger Tan Kay Chia, Prof. Low Kim Huat, Mr Chew Hock See and Dr Mao Shixin (Nanyang Technological University) for their support in setting up and maintaining the research facilities. The authors also thank Prof. Michel M. Maharbiz (University of California, Berkeley) for his advice, Prof. Kris Pister and his group (University of California, Berkeley) for their support in providing the wireless devices used in this study, and Mr Kazuo Unno for providing the photographs of flying insects.