Testing hypotheses of neuromuscular function during locomotion ideally requires the ability to record cellular responses and to stimulate the cells being investigated to observe downstream behaviors []. The inability to stimulate in free flight has been a long-standing hurdle for insect flight studies. The miniaturization of computation and communication technologies has delivered ultra-small, radio-enabled neuromuscular recorders and stimulators for untethered insects []. Published stimulation targets include the areas in brain potentially responsible for pattern generation in locomotion [], the nerve chord for abdominal flexion [], antennal muscles [], and the flight muscles (or their excitatory junctions) []. However, neither fine nor graded control of turning has been demonstrated in free flight, and responses to the stimulation vary widely []. Technological limitations have precluded hypotheses of function validation requiring exogenous stimulation during flight. We investigated the role of a muscle involved in wing articulation during flight in a coleopteran. We set out to identify muscles whose stimulation produced a graded turning in free flight, a feat that would enable fine steering control not previously demonstrated. We anticipated that gradation might arise either as a function of the phase of muscle firing relative to the wing stroke (as in the classic fly b1 muscle [] or the dorsal longitudinal and ventral muscles of moth []), or due to regulated tonic control, in which phase-independent summation of twitch responses produces varying amounts of force delivered to the wing linkages [].

Structure and innervation of the third axillary muscle of Manduca relative to its role in turning flight.

Results and Discussion

th century observations that 3Ax muscle contraction pulls on the 3Ax dorsally and inward, which in these insects tends to pull the wing into a folded posture [ 19 Stellwaag F. Der Flugapparat der Lamellicornier. 20 Straus-Durckheim H. 21 Snodgrass R.E. 14 Balint C.N.

Dickinson M.H. The correlation between wing kinematics and steering muscle activity in the blowfly Calliphora vicina. 17 Rheuben M.B.

Kammer A.E. Structure and innervation of the third axillary muscle of Manduca relative to its role in turning flight. 18 Elson R.

Pflüger H.-J. The activity of a steering muscle in flying locusts. 22 Ando N.

Kanzaki R. Changing motor patterns of the 3rd axillary muscle activities associated with longitudinal control in freely flying hawkmoths. 23 Kammer A.E. The motor output during turning flight in a hawkmoth, Manduca Sexta. Figure 1 Overview of Device-Mounted Beetle and Anatomy of 3Ax Muscle Show full caption (A1 and A2) Overview of the miniature wireless muscular stimulator device (A1); Stimulator device mounted on a live beetle (A2). The device consisted of a custom printed circuit board (PCB) on which a microcontroller, battery with a pair of thin wires, and connector were mounted (see also Figures S4 A and S4B). Four silver wires (127 μm diameter bare, 178 μm diameter Teflon coated) were tightly inserted into the headers, which were mounted on the PCB and electrically connected to the outputs of the micro-processing unit (MPU). The other terminals of the wires were implanted into the left and right wing-folding muscles (3Ax muscle, working electrodes) and the mesothorax center hemolymph (counter electrodes). (B1 and B2) Lateral view of a beetle (B1); close-up view of the red square domain of (B1) after dissection of a cuticle (B2), showing the flight muscle of 3Ax muscle (see also Figure S1 ). Top view of a beetle after the left elytra was removed and the hind wing was unfolded (B1), exposing the left wing base indicated by the red square. (C1 and C2) Close-up view of the red square domain of (C1) to show the 3rd axillary sclerite (3Ax) (C2) that was internally and directly connected to the 3Ax muscle and externally connected to the wing base via a tendon. Figure 2 EMG of 3Ax Muscle Measured during Wing Flapping Followed by Wing Retraction and Free Flight Tests after Various Surgeries Show full caption (A) The 3Ax muscle was activated during the retraction and folding of the wing. Spikes are present not only during the retraction of the wings into a resting position but also while folding wings under elytra. (B) Some cases (66 out of 216, N = 5 animals, n = 216 tests) showed that the 3Ax muscle was not activated during wing retraction and folding. Oscillations in wing tip coordinates indicate flapping. The beetle had one pair of silver wire electrodes implanted into the left 3Ax muscle and a retro-reflective marker attached immediately before the bending zone on each wing at the wing tips; a second marker on the scutellum was used as a reference point. flight = 280 trials, n flap = 270 trials). (C) Severed tendon control for inactivating 3Ax muscle in free flight showed no influence on wing folding/unfolding and flapping but caused the steerage loss. The removal of cuticle had little influence on the free flight since all the beetles (N = 5 beetles, n = 267 trials) flew well at a rate of 97.5% “successful flight” (p < 0.01, binomial test). After 3Ax muscle was cut, all the beetles lost the ability to steer (94.33%, p < 0.01, binomial test) ( Movie S2 ), although they still showed high capability and motivation in flapping wing (89.45%, p > 0.01, binomial test) (N = 5 beetles, n= 280 trials, n= 270 trials). Similar to other insects, Mecynorrhina torquata beetles have a small muscle inserted into the third axillary (3Ax) sclerite, an articulation near the wing base. The 3Ax muscle of the beetle is located between the basalar and subalar muscles ( Figures 1 and S1 ). In beetles, this muscle has often been referred to as the wing-folding muscle, a name dating to 19century observations that 3Ax muscle contraction pulls on the 3Ax dorsally and inward, which in these insects tends to pull the wing into a folded posture []. The 3Ax muscles of other insects are known to play a role in stabilizing flight or steering []. Electromyogram (EMG) recordings of the 3Ax muscle of the beetle show that, although it fires during wing folding ( Figure 2 A), it does not always do so (∼30% or 66 of 216 tests, Figure 2 B); this implies that the 3Ax muscle is not required for wing folding.

The 3Ax muscle is easily isolated and removed in Mecynorrhina ( Figures 1 and S1 ). In all cases (N = 5 beetles, n = 267 trials), removal of the cuticle above the 3Ax muscle did not affect the beetles’ flight, and 3Ax muscle surgery did not prevent beetles from folding or unfolding their wings, nor did it appear to interfere with wing oscillations ( Figure 2 C). However, beetles (N = 5 beetles, n = 280 trials) lost the ability to steer and maneuver in free flight after the 3Ax muscle was isolated and removed ( Movie S2 Figure 2 C). These experiments further imply that the 3Ax muscle is not always required for wing folding but could be involved in flight course control.

24 Rind F.C. A directionally sensitive motion detecting neurone in the brain of a moth. 25 Srinivasan M.

Bernard G. The pursuit response of the housefly and its interaction with the optomotor response. 26 Darwin F.W.

Pringle J.W.S. The physiology of insect fibrillar muscle. I. anatomy and innervation of the basalar muscle of lamellicorn beetles. 27 Burton A.J. Nervous control of flight orientation in a beetle. 28 Burton A.J. Directional change in a flying beetle. 14 Balint C.N.

Dickinson M.H. The correlation between wing kinematics and steering muscle activity in the blowfly Calliphora vicina. 14 Balint C.N.

Dickinson M.H. The correlation between wing kinematics and steering muscle activity in the blowfly Calliphora vicina. 22 Ando N.

Kanzaki R. Changing motor patterns of the 3rd axillary muscle activities associated with longitudinal control in freely flying hawkmoths. 23 Kammer A.E. The motor output during turning flight in a hawkmoth, Manduca Sexta. 18 Elson R.

Pflüger H.-J. The activity of a steering muscle in flying locusts. 18 Elson R.

Pflüger H.-J. The activity of a steering muscle in flying locusts. 22 Ando N.

Kanzaki R. Changing motor patterns of the 3rd axillary muscle activities associated with longitudinal control in freely flying hawkmoths. 23 Kammer A.E. The motor output during turning flight in a hawkmoth, Manduca Sexta. Figure 3 EMG of 3Ax Muscle in Tethered Flight Experiment Show full caption (A) Representative EMG of left 3Ax muscle measured during left and right turns induced by the visual stimulation (projected movie of black and white stripes moving to the left and right, alternately). The 3Ax muscle was activated while the beetle turned to the left. The same tendency was confirmed for the corresponding right side 3Ax muscle. EMG spikes were seen from the left 3Ax muscle during the left turn while no spikes appeared during either right turns or no turns (no moving in the stripes) (and vice versa for the right 3Ax muscle). The green and red bars indicate timings of the visual stimulation for left and right turns, respectively. (B) The spikes appeared during the ipsilateral flow of the pattern at a rate of 83.2% while that of the contralateral one was 3.5% and without stimulation was 13.3%. The error bars represent SD (N = 17 beetles, n = 14,476 spikes). (C–C2) Wing beat trajectories were filmed at 3,000 frame per second and synchronized with EMG of 3Ax muscle, indicating the phase of the wing beat at which each spike occurs. For instance, the spikes indicated by the arrows (C) were fired at the timings of wing beat shown in (C1) and (C2), respectively. (D) Histogram of wing beat phase (0°: beginning of downstroke or end of upstroke; 180°: end of downstroke or beginning of upstroke) at timing when muscle spikes were fired (N = 17 beetles, n = 14,476 spikes/226 bursts). Figure 4 Responses of Beetles to Electrical Stimulation of 3Ax Muscle in Tethered and Free Flight Experiments Show full caption (A1) Displacement of the 3Ax sclerite in response to electrical stimulation of the 3Ax muscle for 500 ms at different stimulus rates (20, 40, 60, 80, and 100 Hz). (A2) Maximum displacement of the 3Ax in response to electrical stimulation of the 3Ax muscle (N = 5 beetles, n = 150 trials). The displacement of 3Ax is normalized by the largest (mechanical limitation of) displacement, which was obtained at the 100-Hz stimulation frequency. The shade area denotes 95% confidence interval. (B) Reduction of the stroke amplitude in response to electrical stimulation of the wing-folding muscle at different rates (60–100 Hz, N = 5 beetles, n = 91 trials). The ipsilateral stroke amplitude was reduced in a graded manner when the wing-folding muscle was stimulated, whereas the contralateral stroke amplitude was retained or fluctuated by an insignificant amount. The shade areas denote 95% confidence interval. (C) Electrical stimulation of the left 3Ax muscle for left turn and the right 3Ax muscle for right turn in sequence produced a zigzag flight path. The black trajectory segments indicate no stimulation periods; red and green trajectory segments indicate right and left stimulation periods, respectively. (D) Lateral force (F l ) induced by the electrical stimulation of 3Ax muscle was graded as a function of stimulus frequency, with the most effective range from 60 Hz to 90 Hz (N right = 12 beetles, n right = 758 trials; N left = 10 beetles, n left = 810 trials). The shade areas denote 95% confidence interval. Values of F l are positive when the direction of force is toward the left and negative when the direction is toward the right. Tonic 3Ax muscle firing occurs during visually induced ipsilateral turns and is correlated with a reduction in ipsilateral wing beat amplitude. In order to examine the behavior of the 3Ax muscle and 3Ax during turns, we unilaterally recorded 3Ax muscle EMGs during visually induced fictive turns in tethered beetles. High-speed videos (3,000 frames per second) were used to map EMG recordings to the stroke cycle and map the wing tip trajectory ( Figures 3 and S2 ). Wide-field optic flow patterns moving either left or right (black and white stripes) produced strong optomotor responses, causing them to turn left or right, respectively, to track optic flow. The latency of optomotor response was found to be 0.81 ± 0.42 s, which is consistent with the slow and variable optomotor responses found in other insects [] (N = 17 beetles, n = 495 tests). All beetles in all cases (N = 17 beetles, n = 226 bursts) activated the 3Ax muscle on the side ipsilateral to the turn, with few spikes occurring in the contralateral 3Ax muscle ( Figures 3 A and 3B). This result is consistent with the hypothesis that the 3Ax muscle plays a role in course corrective turns and demonstrates that the 3Ax muscle is activated during a visually induced ipsilateral turn. In contrast, previously studied coleopteran flight muscles, such as basalar flight muscle, are activated during both ipsilateral and contralateral turns, with the firing rate varying depending on the direction []. Although the 3Ax muscle spikes showed a slight preference for phase, we found they occurred throughout the wing cycle ( Figure S2 ). This is similar to the weak preference found in the tonic steering muscles III1 and I1 in the fly [] and in contrast to the phasic steering muscles found in flies [], moths [], and locusts [], where the muscle shows clear preferred firing phases. The muscles in the latter case produce turning by altering the relative phase between the muscle’s activation and the wing stroke cycle []. Thus, our results are in accord with the suggestion that the beetle performs fine steering not with a single twitch contraction at a specific timing within the stroke cycle but by the summation of multiple twitch contractions (tonic control) that gradates the pull of the wing base. The 3Ax is connected to the wing base via a tendon that allows the 3Ax to transmit force on the wing base ( Figures 1 C and S1 B–S1E). Direct extracellular electrical stimulation of the 3Ax muscle in tethered insects via a micro wire had no influence on the behavior of nearby muscles ( Figure S3 ) but pulled the 3Ax dorsally and inward, as expected. The velocity and displacement of the 3Ax was graded as a function of stimulus frequency ( Figures 4 A1 and 4A2 ; Movie S3 ), suggesting that the 3Ax muscle acts on the 3Ax via graded tonic control arising from the summation of twitch contraction forces.

28 Burton A.J. Directional change in a flying beetle. 29 Taylor G.K. Mechanics and aerodynamics of insect flight control. Turns induced with visual stimuli were associated with either a reduction of the ipsilateral wing stroke amplitude and no change in contralateral wing stroke amplitude, or with no change in the ipsilateral wing stroke amplitude and an increase in the contralateral wing stroke amplitude ( Figures S2 S and S2T) []. An analysis of wing beat trajectory during electrical stimulation of 3Ax muscle shows that 3Ax muscle activation produces a stimulation frequency-dependent reduction of ipsilateral wing stroke amplitude ( Figure 4 B); higher stimulus frequency produced a greater reduction in wing stroke amplitude. This reaction is associated with the correlation of EMG firing rate and wing amplitude change during the fictive turn induced by visual stimulation ( Figure S2 U). In addition, the wing amplitude did not significantly vary as a function of the firing phase ( Figure S2 V).

Remote electrical stimulation of 3Ax muscle enables graded left-right turn control in free flight. Building on these findings, we set out to demonstrate that exogenous stimulation of the 3Ax muscle could produce graded turns in free flight. Our stimulation paradigm was based on three observations: 3Ax muscle firing occurs on the ipsilateral side; firing does not correlate strongly with stroke phase; and reduction of wing stroke amplitude by stimulation of 3Ax muscle is graded with frequency. A radio-enabled backpack ( Figures S4 A and S4B) was mounted on the pronotum, and the left or right 3Ax muscle was stimulated during free flight. Stimulation resulted in clear ipsilateral turns ( Figure 4 C; Movie S1 ). Moreover, the estimated induced lateral force was graded as a function of stimulus frequency ( Figures 4 D and S3 E–S3G; Movie S1 ). The range of the lateral forces induced by the stimulation ( Figure 4 D) was of the same order as those arising from natural (unstimulated) turns ( Figure S3 H).

17 Rheuben M.B.

Kammer A.E. Structure and innervation of the third axillary muscle of Manduca relative to its role in turning flight. 23 Kammer A.E. The motor output during turning flight in a hawkmoth, Manduca Sexta. 17 Rheuben M.B.

Kammer A.E. Structure and innervation of the third axillary muscle of Manduca relative to its role in turning flight. 23 Kammer A.E. The motor output during turning flight in a hawkmoth, Manduca Sexta. 18 Elson R.

Pflüger H.-J. The activity of a steering muscle in flying locusts. 14 Balint C.N.

Dickinson M.H. The correlation between wing kinematics and steering muscle activity in the blowfly Calliphora vicina. 30 Walker S.M.

Thomas A.L.R.

Taylor G.K. Operation of the alula as an indicator of gear change in hoverflies. 31 Nalbach G. The gear change mechanism of the blowfly (Calliphora erythrocephala) in tethered flight. 30 Walker S.M.

Thomas A.L.R.

Taylor G.K. Operation of the alula as an indicator of gear change in hoverflies. 32 Sato, H., Berry, C.W., Casey, B.E., Lavella, G., Yao, Y., VandenBrooks, J.M., and Maharbiz, M.M. (2008). A cyborg beetle: insect flight control through an implantable, tetherless microsystem. IEEE 21st International Conference on Micro Electro Mechanical Systems, 2008. MEMS 2008, 164–167. 33 Sato, H., Peeri, Y., Baghoomian, E., Berry, C.W., and Maharbiz, M.M. (2009). Radio-Controlled Cyborg Beetles: A Radio-Frequency System for Insect Neural Flight Control. IEEE 22nd International Conference on Micro Electro Mechanical Systems, 2009. MEMS 2009. 216–219. 34 Dickinson M. Insect flight. 14 Balint C.N.

Dickinson M.H. The correlation between wing kinematics and steering muscle activity in the blowfly Calliphora vicina. 35 Forbes W.T. The wing folding patterns of the Coleoptera. While we know of no previous reports detailing a flight function for this muscle in coleopterans, a comparison of these findings with those in moths, locusts, and flies seems to imply a previously unappreciated similarity in function between the coleopteran 3Ax muscle and the muscles that insert into the 3Ax in other insects. In moths, this muscle is active during straight flight and is phase shifted with regards to the dorsal longitudinal muscle during maneuvers []. Furthermore, the tonic activation of this muscle maintained a certain degree of remotion and elucidated wing retraction in moth []. In locusts, the similar muscle of the forewing has been shown to play a central role in steering and is part of the visual control system. It is activated on both sides during straight flight, and turns are correlated with both phase shifts from the baseline and changes in spike frequency []. In contrast, in dipterans (notably Calliphora and Eristalis), the 3Ax has been implicated in the control of the alula, a hinged flap present near the base of dipteran wings []. The alula accounts for a small percentage of fly wing area and is held flat or raised during flight bouts. Engagement of the alula is correlated to switching flight modes (or “gear shifting”) during flight. Moreover, dipterans are capable of operating the gear change mechanism associated with flipping their alula asymmetrically to reduce aerodynamic force, resulting in ipsilateral turning []. In addition, the I1 muscle of fly was activated with change of firing rate in a weak phasic manner also associated with the reduction of stroke amplitude that would induce ipsilateral turn []. Coleopterans lack an alula, although the wings contain a similar fold near the base []. However, our results show that the 3Ax muscle tonically contracts (on the side ipsilateral to a turn) or does not contract (on the side contralateral to a turn), which is reminiscent of the muscles on the dipteran 3Ax. It is tempting to speculate that coleopterans possess a similar “asymmetrically switchable” muscle even lacking the clear “gear shifting” structures present in dipterans.