Resilin and chitinous cuticle form a composite structure for energy storage in jumping by froghopper insects

Abstract

Background: Many insects jump by storing and releasing energy in elastic structures within their bodies. This allows them to release large amounts of energy in a very short time to jump at very high speeds. The fastest of the insect jumpers, the froghopper, uses a catapult-like elastic mechanism to achieve their jumping prowess in which energy, generated by the slow contraction of muscles, is released suddenly to power rapid and synchronous movements of the hind legs. How is this energy stored?

Results: The hind coxae of the froghopper are linked to the hinges of the ipsilateral hind wings by pleural arches, complex bow-shaped internal skeletal structures. They are built of chitinous cuticle and the rubber-like protein, resilin, which fluoresces bright blue when illuminated with ultra-violet light. The ventral and posterior end of this fluorescent region forms the thoracic part of the pivot with a hind coxa. No other structures in the thorax or hind legs show this blue fluorescence and it is not found in larvae which do not jump. Stimulating one trochanteral depressor muscle in a pattern that simulates its normal action, results in a distortion and forward movement of the posterior part of a pleural arch by 40 microm, but in natural jumping, the movement is at least 100 microm.

Conclusion: Calculations showed that the resilin itself could only store 1% to 2% of the energy required for jumping. The stiffer cuticular parts of the pleural arches could, however, easily meet all the energy storage needs. The composite structure therefore, combines the stiffness of the chitinous cuticle with the elasticity of resilin. Muscle contractions bend the chitinous cuticle with little deformation and therefore, store the energy needed for jumping, while the resilin rapidly returns its stored energy and thus restores the body to its original shape after a jump and allows repeated jumping.

Figure 1

Structure of the internal skeleton of the metathorax of Aphrophora. (A) Ventral view of the left half of the metathorax and proximal joints of the left hind leg. The box on the inset drawing indicates the area drawn in detail. The ventral cuticle of the thorax (orange) and the muscles were removed to reveal the pleural arch (red), linking the lateral edge of the coxa of the left hind leg (green) ventrally to the articulation of the left hind wing dorsally. (B) Dorsal view to show the left pleural arch curving ventrally from the hinge of the left hind wing to the lateral hinge of the thoraco-coxal joint of the left hind leg. The large tendon of the trochanteral depressor muscle is also shown.

Figure 2

Fluorescent structures in the metathorax of Aphrophora and Philaenus. Externally visible fluorescence at the right (A) and left (B) lateral pivot of the thoraco-coxal joints of the hind legs of Aphrophora. Bright field images and those illuminated with ultraviolet light (see Methods) at the same position and focal plane are superimposed. (C) Ventral view of the internal metathorax of Philaenus after removal of the ventral cuticle and thoracic muscles. The pleural arches linking the coxae with the hinges of the hind wings are outlined with dashed lines. Part of each pleural arch fluoresces bright blue.

Figure 3

Intense blue fluorescence in part of the pleural arch of the left half of the metathorax of Aphrophora. (A) Ventral view. The ventral cuticle and muscles of the left metathorax were removed, but the tracheae and some fatty tissue remain medially. (B) Medial view looking outwards after the metathorax had been split at the midline and muscles removed. (C) Lateral view from a montage of photographs. The lateral wall and muscles of the metathorax were removed. The dashed lines indicate the outline of the whole pleural arch. The fluorescent part is clearly curved antero-posteriorly (A and C) and dorso-ventrally (B and C).

Figure 4

Development of fluorescence during maturation of successive larval stages of Philaenus. (A)and (B) In two larvae with body lengths of 4.1 mm and 4.8 mm, there was no fluorescence at the thoraco-coxal joints as there was in adults (diagonal yellow arrows). Some fluorescence is apparent in the soft membrane between the proximal joints of the hind legs and in the mouthparts (horizontal black arrow in A). (C) A larger larva (5.2 mm body length) shows strong fluorescence at the thoraco-coxal joint of the left but little at the right hind leg. (D) In the larval stage (6.7 mm long) preceding the final moult to adulthood, fluorescence is present at both thoraco-coxal joints. In (A), the two hind legs are photographed in one image, in (B)-(D), they are represented in two images. The dotted lines indicate the midline.

Figure 5

Changes in the intensity of fluorescence with pH. (A) Side view of the right half of the metathorax after the cuticular side wall and thoracic muscles were removed; the original colour images, taken at the times indicated, have been converted to black and white. The insect was initially in saline at pH 7.2 and at time 0 minutes this was replaced by saline at pH 2 and after 13.5 minutes by saline at pH 12. In the acid pH, the intensity of the fluorescence gradually declined, only to increase again in the alkaline pH. (B) Graph of the changes in intensity measured from digital images, such as those shown in (A). Red squares and lines: summed fluorescence across pixels, normalised, within an outline approximately 1 mm long drawn to cover the brightest part of the pleural arch. Black filled circles: normalised mean maximum fluorescence intensity ± 1 standard deviation recorded along 10 lines approximately 0.55 mm long and 0.1 mm apart, orthogonal to the long axis of the pleural arch. Blue circles: background fluorescence assayed as the mean minimum intensity ± 1 standard deviation from the same lines.

Figure 6

Partial emission spectrum of the fluorescence. The pleural arches were measured in six Aphrophora (filled squares ± 1 standard deviation) using five accessory interference filters with peaks falling within the 413–483 nm pass band of the Semrock emission filter. These measurements are compared with the known emission spectra of native resilin (diamonds, from Andersen [25]) and synthesised dityrosine (filled circles, from Malencik et al. [27]).

Figure 7

Distortion of the pleural arches in jumping. (A) Movement of the coxal end (see box in right image of B) of the pleural arch of the left hind leg when the left trochanteral depressor muscle was stimulated. Digital images under ultraviolet illumination at the start (cyan) and end (magenta) of the stimulation are superimposed. The colour changes were made in Photoshop, so that areas of overlap between the two images appear white. (B) Frames from a natural jump captured at 1000/second; the left frame was taken before the jump, the middle one after a 2-second long contraction of the trochanteral muscles, and the one on the right after the jump. The prolonged contraction results in a forward movement of the coxae (arrow and lines), which is reversed when the hind legs depress and extend in a jump.