Sophisticated suction organs from insects living in raging torrents: Morphology and ultrastructure of the attachment devices of net-winged midge larvae (Diptera: Blephariceridae)

Suction organs provide powerful yet dynamic attachments for many aquatic animals, including octopus, squid, remora, and clingfish. While the functional morphology of suction organs from various cephalopods and fishes has been investigated in detail, there are only few studies on such attachment devices in insects. Here we characterise the morphology, ultrastructure, and in vivo movements of the suction attachment organs of net-winged midge larvae (genus Liponeura) – aquatic insects that live on rocks in rapid alpine waterways where flow rates can reach 3 m s-1 – using scanning electron microscopy, laser confocal scanning microscopy, and X-ray computed micro-tomography (micro-CT). We identified structural adaptations important for the function of the suction attachment organs from L. cinerascens and L. cordata. First, a dense array of spine-like microtrichia covering each suction disc comes into contact with the substrate upon attachment. Similar hairy structures have been found on the contact zones of suction organs from octopus, clingfish, and remora fish. These structures are thought to contribute to the seal and to provide increased shear force resistance in high-drag environments. Second, specialised rim microtrichia at the suction disc periphery form a continuous ring in close contact with a surface and may serve as a seal on a variety of surfaces. Third, a V-shaped cut on the suction disc (the V-notch) is actively peeled open via two cuticular apodemes inserting into its flanks. The apodemes are attached to dedicated V-notch opening muscles, thereby providing a unique detachment mechanism. The complex cuticular design of the suction organs, along with specialised muscles that attach to them, allows blepharicerid larvae to generate powerful attachments which can withstand strong hydrodynamic forces and quickly detach for locomotion. Our findings could be applied to bio-inspired attachment devices that perform well on a wide range of surfaces.


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The suction disc rim was not the only region of the suction disc that came into close contact

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There is a gradual transition from the flat rim microtrichia to the longer spine-like microtrichia.

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While the morphology of the short rim microtrichia differs between the two Liponeura species,

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In addition to the rim and spine-like microtrichia, we observed fine structures near the central 206 opening also making contact (Fig. 4). These fine central microtrichia are much shorter than 207 the spine-like microtrichia, and lack the pitch and curvature (Fig. 5b). We observed that 208 when the piston was retracted (lowering the internal pressure), the central area was brought 209 closer to the surface and a larger area of the central microtrichia zone came into contact At the anterior side of each suction disc, the V-notch sharply disrupts the circular outline of 224 the disc in both Liponeura species (Fig. 5a and Fig. 6). When the V-notch is shut, the sealing 225 rim is almost continuous (Fig. 4), but when open at its widest aperture, the V-notch gap is 226 2.6 ± 0.3% (n = 3, L. cinerascens) of the perimeter of the disc (i.e., for a fourth instar larva 227 with 440 µm disc diameter, the V-notch is around 36 µm wide). The V-notch extended from 228 the edge until the end of the spine-like microtrichia zone (33.3 ± 2.7% of the suction disc 229 radius, from n = 3, L. cinerascens) and the area surrounding the base of the V-notch lacked 230 spine-like microtrichia. Through in vivo IRM recordings, we clearly observed local, active 231 movements of the V-notch: when the suction disc was firmly attached to a surface, the V-232 notch was closed, but prior to detachment during locomotion, the two lateral flanks of the V-

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notch were peeled open starting from the outer end, thereby breaking the seal and rapidly 234 equalising the pressure (Fig. 6a, and supplementary materials SV5). We also observed (n = 1 larva). The V-notch apodemes did not appear to be attached to the cuff wall or to any 247 surface as they circumvented around the cuff and into the body. The cross-sectional area of 248 the V-notch muscles were smaller than other muscle groups associated with the suction 249 organ (piston, cuff, cuticle fold muscles; see Table 1

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In addition to the apodemes and muscles of the V-notch, a second novel feature of the V-269 notch was identified: in a sagittal view rendering from micro-CT data, it can be seen that the 270 V-notch is shaped like a cupped hand and distinctly juts out further dorsally than the 271 posterior side of the suction disc that has no V-notch (Fig. 8). This membranous structure 272 may have a valve-like function, as it could be seen widening and fluttering as the piston was 273 lowered and water expelled through the valve, but when the piston was raised and the 274 internal pressure lowered, the valve stopped moving (supplementary video SV6). It is likely 275 that this structure and the associated movements serve to seal the V-notch, as proposed a single microtrichium (shaded in red) to highlight the curvature and pitch angle (see Table 1