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First published online June 7, 2004
Journal of Experimental Biology 207, 2455-2464 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.01039

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Cardiac-like behavior of an insect flight muscle

Michael S. Tu^* and Thomas L. Daniel

Department of Biology, University of Washington, Seattle, WA 98195-1800, USA

View larger version (39K): [in a new window]   Fig. 1. Preparation for measurement of muscle length changes during tethered flight and length–tension measurements in the intact thorax. (A) Lateral view of Manduca showing placement of length transducer probes (p) and optical sensors (os) used to measure length changes of the dl1 muscles during tethered flight. (B) The five sub-units of each dl1 muscle (a–e) attach to two invaginations of the exoskeleton, the 1st phragma anteriorly and the 2nd phragma posteriorly. Acting indirectly through a complex wing articulation, contraction of the dl1 muscles depresses the wings. The probes were inserted through incisions in membranous areas of the abdomen and neck and hooked onto the 1st and 2nd phragmata. The moth was held on a brass rod (br) glued between the bases of the mesothoracic legs. We define the anatomical rest length, Lr, as the length of dl1a along its ventral surface in the intact thorax of a quiescent moth. In practice, we measured Lr as the distance separating the hooks of the displacement transducer probes with the moth at rest. For isometric twitch length–tension measurements in the intact thorax, the probe hooks were bilaterally paired rather than single, but the method of insertion and placement was the same. The anterior probe was rigidly fixed in place, and the posterior probe was connected to an isometric force transducer. s, silhouette of body scales.

View larger version (25K): [in a new window]   Fig. 2. Preparation used to measure isometric twitch forces from mechanically isolated dl1 muscles. (A) Placement of the anterior (a) and posterior (p) muscle grips for twitch force measurements on mechanically isolated muscles. (B) After decapitating the moth, the 1st phragma was exposed and the anterior grip placed over the anterior insertion of the dl1 muscles. The paired needles of the posterior grip were driven through the dorsal cuticle and down along the posterior face of the 2nd phragma. Both grips were secured to the exoskeleton with cyanoacrylate adhesive. After gluing acetate strips (not shown) across the gap between the two grips to fix their relative positions, cuticle strips were excised (arrows, broken line) to mechanically isolate the anterior grip and muscle insertions from the rest of the thorax. The anterior grip was then secured to the force transducer via the threaded rod projecting from the grip. The ball bearing (bb) mounted on the posterior grip fit into a depression in the end of a threaded rod (tr) mounted on a translation stage. When secured by a slotted retaining nut (n), the ball bearing and threaded rod formed a ball joint. The acetate strips spanning the two grips were then cut, and any misalignment of the cut ends was corrected using the ball joint and translation stage.

View larger version (12K): [in a new window]   Fig. 3. (A) Strain and (B) electromyogram (EMG) signals recorded from the dl1 muscle during tethered flight. In the sequence shown, the rest length in the quiescent moth (Lr; upper broken line) is 4% longer than the operational length (Lop; lower broken line). The dl1 muscles fired a single spike near the onset of muscle shortening in each wing-stroke. The time scale (ordinate) is the same for both traces. The wing-stroke frequency in this sequence was 23 Hz (Table 1, moth 4).

View larger version (14K): [in a new window]   Fig. 4. Superimposed Fourier transforms of the strain records from six moths. For each flight sequence, the frequency values (abscissa) have been normalized by dividing the frequency of the first (dominant) Fourier coefficient by the wing-beat frequency of that trial. The mean distortion of the strain trajectories from sinusoidal was 25±17% as measured by the ratio of the dominant coefficient to that of the first harmonic.

View larger version (19K): [in a new window]   Fig. 5. Isometric twitch length–tension curves derived from active twitch forces measured from the dl1 muscles. The dl1 muscles operate exclusively on the ascending limb of their length–tension curve. (A) Isometric twitch length–tension curve based on in situ measurements from the intact thoraces of seven moths. (B) Isometric twitch length–tension curves measured from mechanically isolated dl1 muscles from four moths. (C) Length–tension curves representing pooled data from intact thorax preparations (heavy line) and from isolated muscle preparations (light line). All curves in A and B are 2nd order polynomials fit to individual data sets. Each curve in C was obtained by averaging the polynomial coefficients from the individual curves within each of the two data sets shown in A and B. The solid vertical line in each plot indicates the mean value of operational length (Lop), measured relative to the anatomical rest length of the dl1 muscles in six tethered flight preparations. The broken vertical lines (a, mean minimum muscle length; b, mean maximum muscle length) indicate the average bounds of muscle length changes during tethered flight. Muscle length for active twitch force measurements in the intact thorax preparations (A) was also referenced to the anatomical rest length. This common length reference allowed us to map the in vivo muscle length changes measured in tethered flight onto the twitch length–tension curve measured in intact thorax preparations (A). The vertical lines indicating Lop and the mean range of length changes derived from measurements in tethered flight and from the intact thorax preparation are replicated in B and C. Although the intact thorax and isolated muscle preparations produced different curves, both indicate that active twitch force is substantially lower than maximal over the range of in vivo muscle lengths.

View larger version (12K): [in a new window]   Fig. 6. Active twitch length–tension curve for the dl1 muscles of Manduca (light solid line), mammalian cardiac muscle (heavy solid line; redrawn from Allen and Kentish, 1985), cat soleus muscle (light broken line; redrawn from Rack and Westbury, 1969) and the length–tension curve for tetanized cat soleus muscle (heavy broken line; redrawn from Rack and Westbury, 1969). Compared with mammalian skeletal muscle, the dl1 muscles and mammalian cardiac muscle both have length–tension relationships with steep ascending regions and narrow peaks, even compared with mammalian skeletal muscle at low levels of activation. The curve for Manduca dl1 muscles is the same as that shown for mechanically isolated muscles in Fig. 5C.

View larger version (15K): [in a new window]   Fig. 7. The time course of a twitch force depends upon where along the length–tension curve the muscle operates. The twitch force (Ft) was modeled with the function Ft=exp[–1000(t–0.01)2/t0.5], where t is time in seconds. Force is plotted against time with (a) no length dependence of the force, (b) a length dependence of force that increases linearly in time with a strain of 0.5 [Ft(1– t/0.04); e.g. contraction on the ascending portion of the length–tension relationship] and (c) a length dependence that decreases linearly in time with the same strain as in b [Ft(1+ t/0.04–); e.g. contraction on the descending portion of the length–tension relationship]. Operating along the ascending portion leads to an earlier peak and a more rapid decline in force.