H. lugubris larvae have six ventromedian suction organs, with each organ comprising a suction disc, a central opening and a piston, a suction chamber surrounded by a thick-walled cuticular cuff, and a V-notch (Figure 1c-f and Video 2). The suction disc contacts the surface for attachment, and the piston and underlying piston muscles (Figure 1d & e) actively lower the pressure inside the suction chamber. Two apodemes attaching to the V-notch in H. lugubris mediate its muscle-controlled opening for rapid detachment of the suction organ (Figure 1f; see also Kang et al., 2019).

Video 2

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The ventral disc surface of H. lugubris is covered in a dense array of microtrichia (Figure 2a-e). The suction disc-sealing rim, which seals the disc for suction attachment, closely resembles that of Liponeura cinerascens (Kang et al., 2019) and comprises a dense array of upright rim microtrichia (Figure 2b). This is different from Liponeura cordata, which has a distinct rim made up of a single row of horizontally flat rim microtrichia (Kang et al., 2019). Going from the rim to the centre of the disc, the short rim microtrichia transition into longer spine-like microtrichia (6.7 ± 0.5 µm in length and 0.56 ± 0.01 µm in mid-length diameter; mean of means ± standard error of the mean; measured from scanning electron microscopy (SEM) images of n = 2 individuals), and then again to shorter microtrichia in the centre.

Figure 2

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The following imaging techniques were used to gain insights into the ultrastructure and internal organisation of the suction disc: freeze-fracture SEM, 3D models using computed microtomography (micro-CT) data, and in vivo transmitted light microscopy (Figure 2). While internal fan-fibre networks underneath the outer regions of the suction disc have been mentioned previously (Rietschel, 1961), we discovered that each internal fibre leads to a single microtrichium (Figure 2b-d). Moreover, all the microtrichia that were fractured during sample preparation appeared to be solid (in-filled) cuticular structures (Figure 2e). The small internal fibres leading into the microtrichia branch out from thicker trunks originating from the ventral side of the outer radial beams (Figure 2b-e). Another notable ultrastructural feature is the radial beams, which are solid cuticular structures that alternate between a wide and a narrow beam (Figure 2c). The beams originate from the palisades, a radial zone consisting of dorsoventral cuticular rods (Figure 2f & g). There are 72 radial beams in a 90° segment of the disc, corresponding to 288 beams per disc (assuming no interruption from the V-notch) and a centre-to-centre spacing of around 4 µm or 1.3°.

Blepharicerid larvae possess some of the most powerful (in terms of body weight) and complex suction organs among animals. The three species of blepharicerid larvae studied here (H. lugubris, L. cordata, and L. cinerascens) produced extreme shear forces on smooth surfaces with averages that ranged from 320 to 1120 times their own body weight. In terms of weight-specific attachment performance, the larvae performed better than all terrestrial insects measured using comparable methods (ie, whole-animal detachment experiments) (Federle et al., 2000; Grohmann et al., 2014). For example, the weight-specific shear attachment of blepharicerid larvae on smooth surfaces was 3–11 times greater than that of stick insects measured in this study. To achieve this extreme shear attachment, blepharicerid suction organs must come in close contact by generating an effective seal. Based on our in vivo visualisations of H. lugubris attaching to smooth glass underwater, the microtrichia make close contact with the surface, helping to both seal the organ and generate friction. This corroborates our previous findings on the suction disc contact behaviour with L. cinerascens and L. cordata (Kang et al., 2019). Likewise, the soft adhesive pads of stick insects make close contact on smooth surfaces, and while the weight-specific attachment forces are not as high as in blepharicerid larvae, they can withstand forces close to 100 times their body weight.

In contrast, the attachment of stick insects on micro-rough surfaces is significantly different to that of blepharicerid larvae: for stick insects, there was a 16-fold decrease in performance compared to smooth substrates, while for blepharicerid larvae, the decrease was only twofold. This difference in the impact of micro-roughness can be attributed to the two fundamentally different mechanisms of attachment: on micro-rough surfaces, neither the soft adhesive pads nor the tarsal claws of stick insects function properly (Bullock and Federle, 2011). This is in part due to the reduced effective contact area (the adhesive pads cannot mould sufficiently to the asperities) and also due to the reduced friction from tarsal claws (the claws cannot interlock with the small asperities). On the other hand, blepharicerid suction organs are still able to seal on micro-rough surfaces and microtrichia can interact with the asperities, which likely explains why their performance was not as diminished as in the stick insects. In addition, blepharicerid suction organs may adhere better to micro-rough surfaces because during partial contact, the gaps between the detached regions and the substrate are filled with water, whereas detached regions of stick insect pads are filled with air. As water is effectively incompressible and approximately 50 times more viscous than air, the water-filled contact zone can provide a much stronger resistance against detachment even under conditions of partial contact as on micro-rough substrates.

While blepharicerid larvae attached more strongly than stick insects on micro-rough surfaces, the opposite was found on coarse-rough surfaces: for stick insects, there was no difference in performance between coarse-rough and smooth surfaces, whereas blepharicerid larvae attachment decreased 11-fold. It is likely that both blepharicerid suction organs and stick insect adhesive pads are unable to cope with coarse surface roughness. The adhesive pads of both insects may be unable to fully mould to the large asperities, and the length of the microtrichia may be insufficient to reach the lower regions of the surface profile (Figure 6). Stick insects, however, have large pretarsal claws that can interlock with large asperities for strong attachment. Previous studies on dock beetles (Gastrophysa viridula) and stick insects (C. morosus) have reported that both beetle and stick insect attachments on coarse-rough surfaces decrease significantly when the claws are removed (Bullock and Federle, 2011; Scholz et al., 2010). This means that, although stick insects and dock beetles use two distinct adhesive systems (smooth versus hairy pads), the combination of the claws and the adhesive pads produces the same trend: both insects attach strongly to smooth and coarse-rough surfaces but poorly to micro-rough surfaces. In contrast, blepharicerid larvae do not have claw-like appendages and rely on suction organs for attachment. Consequently, these aquatic larvae do not follow the same trend as terrestrial climbing insects and perform the worst on coarse-rough substrates. A similar result was reported by Liu et al., 2020 using Blepharicera sp., where the larval attachment performance decreased with increasing surface roughness, although no quantitative information on attachment forces can be extracted from their study (the study also used a centrifuge, but only reported the rotation speed but not the insects’ mass and position; it is also unclear whether the larvae were wetted prior to the tests).

Although attachment forces per body weight help to assess the performance from a biological perspective, the attachment force per contact area, or the stress, is needed to make comparisons between animals that adhere to surfaces. In blepharicerid larvae, the shear stress on smooth substrates ranged from 39 to 117 kPa, and from 24 to 120 kPa for normal stress (the range in values arises from the assumptions used to calculate the contact area and the number of organs remaining in contact immediately prior to detachment). These values are similar to those reported in the literature for suction attachments of other animals on smooth substrates: the remora fish can withstand 93 kPa in shear and 38 kPa in the normal direction (Fulcher and Motta, 2006); octopus can resist normal stresses of up to 271 kPa, squids up to 830 kPa, and lumpsucker fish up to 102 kPa (Smith, 1996; Davenport and Thorsteinsson, 1990). Like the cephalopods, blepharicerid suction stress can surpass 101 kPa (1 atm at standard sea level and temperature), with one L. cordata reaching 228 kPa (realistic estimate; 77 kPa if based on conservative assumptions, as outlined in ‘Results’ section). Although a pressure difference of 1 atm is considered the upper threshold for suction attachments in air, this is not the case if the contact zone is completely wet and bubble-free, as the strong cohesion of water allows suction stresses to exceed 1 atm (Smith, 1991; Smith, 1996; Wang et al., 2019; Wang et al., 2020). Even if they do not reach 1 atm, blepharicerid larvae can generate sufficient attachment force to resist the fast flow rates in their natural habitat.

Recent work on cupped microstructures (which resemble microscopic suction cups) has revealed the mechanisms of failure in underwater suction attachments: (1) under sustained tensile stress, the rim slides inwards and the rim diameter contracts by ~30%; (2) immediately prior to detachment, sections of the rim buckle inwards, leading to adhesive failure (Wang et al., 2019). Similar failure modes were also reported for macroscopic suction cups (Ditsche and Summers, 2019). Two structural features of the blepharicerid suction organs could represent adaptations to counter the aforementioned failure mechanisms seen in cupped microstructures. First, the internal radial beams can provide structural support to reduce inward sliding and buckling of the suction disc rim. Similar to how the flexible membrane of an umbrella is stiffened by radial spokes, these stiff cuticular radial beams can stabilise the suction disc when the organ is under high tensile stress. Bones within the clingfish suction organ may also prevent inward sliding (Wang et al., 2020). While we have yet to visualise blepharicerid suction organs fail under extreme forces, their powerful attachments suggest that they possess mechanisms to counter suction cup failure.

The second morphological feature that may reduce inward sliding of the rim is the numerous microtrichia in the contact zone of the blepharicerid suction disc. The microtrichia can interlock with surface asperities and minimise inward sliding. This has been reported for the remora fish, which have stiff posterior-facing structures called spinules within their suction pads that passively engage with asperities during high-drag conditions (Beckert et al., 2015; Fulcher and Motta, 2006), as well as for the clingfish, where a hierarchical system of rods and filaments near the periphery of the suction organ may increase friction on rough surfaces (Ditsche and Summers, 2019). Similarly, blepharicerid microtrichia are naturally angled (~40° to 50° relative to the horizontal) and point towards the centre of the suction disc (Kang et al., 2019). Hence, inward sliding would passively lead to additional interlocking of the microtrichia tips with rough surfaces, thereby loading the microtrichia along their axis.

To interlock effectively, structures like the remora spinules need to be stiff and strong (Dai et al., 2002; Wang et al., 2017). The blepharicerid microtrichia are indeed likely to be stiff structures since (1) they are made of solid cuticles (a composite material of chitin fibres embedded in a matrix of proteins), and the dense, sclerotised cuticle can reach high elastic moduli [Parle et al., 2017]; (2) we never observed any microtrichia in side contacts, even on micro-structured surfaces when the disc was pressed into contact. From the observation that microtrichia only make tip contact, and assuming that they are loaded equally, we can give a conservative lower estimate for the elastic modulus of the microtrichia cuticle by following Goss and Chaouki, 2016 and modelling the microtrichium as a cylindrical beam loaded at an angle (Figure 7; see ‘Materials and methods’ section for details). Based on this model, the microtrichia cuticle must have a stiffness of at least 0.3–0.4 GPa, similar to the stiffness of wood and bone (Vincent and Wegst, 2004), to prevent any side contact (for an angle to the disc surface of 40° to 50°; see Figure 7). It is not unlikely that the microtrichia cuticle is even stiffer: sclerotised cuticles can have an elastic modulus of up to 20 GPa (Vincent and Wegst, 2004; Wegst and Ashby, 2004; Sykes et al., 2019). This supports our idea that microtrichia can maintain tip contact during interactions with rough surfaces and could serve a similar function to stiff remora spinules.

Figure 7

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It is worth mentioning that, while stiff interlocking structures play an important role in increasing friction on rough surfaces, an effective seal is crucial for attachments on both smooth and rough surfaces. In remora fish and clingfish, a soft rim helps the organ to mould to the surface and form a seal (Wang et al., 2020). In bio-inspired suction devices as well, it is the combination of stiff structures and a soft sealing rim that generates the strongest attachments to smooth and rough surfaces (Ditsche and Summers, 2019; Wang et al., 2017). The detailed biomechanics of how the dense array of microtrichia produces a tight seal on rough surfaces and interlocks with small substrate asperities is beyond the scope of this study and remains to be explored in future work.

We have demonstrated that blepharicerid suction organs can attach with extreme strength to both smooth and rough surfaces. Despite the potential strength of each attachment, the larvae are surprisingly mobile in their natural habitat (Frutiger, 1998; Federle and Labonte, 2019). Both attachment force and mobility are required for blepharicerid larvae to survive in their challenging habitats, which include raging alpine torrents and areas near the base of waterfalls. Currently, blepharicerid suction organs are the only examples of piston-driven suction organs in insects. While the circular setae of male diving beetles (Dytiscidae) are also considered to be suction organs, the two systems have markedly different morphologies: (1) the ventral surfaces of circular setae are comparatively smooth; (2) circular setae lack a muscle-driven central piston; (3) there are no known mechanisms for rapid detachment in dytiscid circular setae (Chen et al., 2014; Karlsson Green et al., 2013; Nachtigall, 1974). As male dytiscid beetles use their suction organs to attach to the smooth sections of the female’s pronotum and elytra, their attachment works best on smooth surfaces and their performance declines strongly on rough surfaces (Karlsson Green et al., 2013). Researchers have suggested that male and female dytiscid beetles are engaged in an evolutionary arms race driven by sexual conflict, as female cuticular surfaces are modified to hinder male attachment with suction discs (Karlsson Green et al., 2013; Bergsten et al., 2001). Interestingly, it appears that male beetles did not evolve friction-enhancing structures on their circular setae to facilitate adhesion to rougher regions of the female cuticle. In contrast, blepharicerid suction discs are densely covered in microtrichia that likely enhance the grip on rough surfaces in high-drag conditions. This difference in morphology may be based on function or phylogenetic constraints: male diving beetles may not need to attach to rough elytra if their setae can generate sufficient attachment forces on smooth cuticle alone; alternatively, beetle setae may be more limited in the structures that can be developed from them, compared to the blepharicerid organ, which is highly complex and multicellular.

A more comparable suction-based attachment system can be found in remora fish. Remora fish use suction pads—highly modified dorsal fin spines—to attach to sharks, whales, and manta rays (Beckert et al., 2015; Fulcher and Motta, 2006). Recent studies on the functional morphology of remora suction pads have greatly expanded our understanding on the mechanisms underlying their impressive performance (Beckert et al., 2015; Wang et al., 2017; Gamel et al., 2019). A remora suction pad comprises a soft fleshy outer rim and rows of lamellae topped with spinules. The pitch of the lamellae is muscle-controlled to facilitate spinule contact with rough surfaces, such as shark skin. When engaged, the tips of these stiff spinules interlock with surface asperities and increase friction, thereby increasing shear resistance. Moreover, the angled posterior-facing lamellae and spinules promote passive engagement when subjected to shear forces from a swimming host (Beckert et al., 2015; Fulcher and Motta, 2006). The similarities between the remora suction pad and the blepharicerid suction organ may be based on overlapping functional requirements, as both animals have to cope with high shear forces (fast-swimming hosts for the remora and torrential rivers for blepharicerid larvae).

Since blepharicerid larvae attach to rocks underwater and feed on epilithic algae, their suction organs will in most cases contact the biofilm, yet the details of this interaction are unknown (Frutiger and Buergisser, 2002). As hypothesised previously, it is possible that the stiff microtrichia penetrate the biofilm layer (Rietschel, 1961; Nachtigall, 1974). This may allow the microtrichia to directly interlock with asperities on the rock surface or to generate additional friction from embedding numerous microtrichia into the biofilm. Mayfly larvae, which also inhabit fast-flowing watercourses, have friction-enhancing hairs that benefit from interacting with the biofilm (Ditsche et al., 2014a). It was found that a higher proportion of mayfly larvae can withstand fast flow rates on smooth hard (epoxy) substrates when the biofilm is present. Moreover, the setae and spine-like acanthae on the ventral surfaces of mayfly larvae can generate friction forces on clean rough substrates (Ditsche-Kuru et al., 2010). Additional experiments with blepharicerid larvae are underway to investigate the interaction between stiff microtrichia and soft substrates.

To conclude, we have shown that blepharicerid larvae use their suction organs to generate extreme attachment to diverse surfaces. The suction organ morphology is conserved between Hapalothrix and Liponeura larvae, and consists of a suction disc that contacts the substrate, dense arrays of microtrichia on the disc surface, muscles to control the piston, and the V-notch detachment system. We characterised the suction disc ultrastructure, which includes internal radial beam structures that could help to stabilise the suction disc when subjected to high stress,and fan-fibres that connect individual microtrichia to the radial beams. In terms of attachment performance, blepharicerid larvae withstand extreme shear forces equivalent to 320–1120 times their body weight on smooth substrates, depending on the species. H. lugubris performed the best overall, reaching shear forces equivalent to 1430 times the body weight, as well as an estimated shear stress of 117 kPa and normal stress of 24 kPa. Although their attachment decreased with increasing surface roughness, blepharicerid suction organs performed better than the smooth adhesive pads of stick insects on micro-rough surfaces. We confirmed that blepharicerid suction organs can mould to large surface asperities and that microtrichia come into close contact between the asperities. These microtrichia are stiff spine-like structures that are specialised for maintaining tip contact with the surface for interlocking with asperities. Our study provides new insights into the function of a highly adapted insect adhesive organ and expands our understanding of the function of biological suction organs.

L. cinerascens (Loew, 1845) larvae were collected from fast-flowing alpine rivers near Meiringen, Switzerland (GPS location 46° 44' 05.6" N, 8° 06' 55.4" E, in May 2018), and close to Grinzens, Tirol, Austria (47° 12' 41.4" N, 11° 15' 28.1" E, in September 2018). At the latter site, L. cordata (Vimmer, 1916) and H. lugubris (Loew, 1876) were also collected. For all the species, we collected third and fourth instar larvae that were large enough to be handled for experiments. Wearing fishing waders and diving gloves, we removed rocks from the most turbulent areas of the river and brought them to the riverbank for specimen collection. Although it was previously noted that the larvae can attach so firmly that they are torn upon detachment (Komárek, 1914), we found that a gentle nudge using soft-touch tweezers can elicit an evasive response from them, whereupon they could be easily picked up using tweezers and placed in specially prepared 50 ml Falcon tubes. All larvae were kept in these tubes in an ice box during collection and transport. Rocks were returned to their approximate locations after collection.

For long-term maintenance of the larvae, an aquarium tank was set up with water and small rocks from the collection site. A filter unit with two outlets for a small water cascade was used to filter the water and to simulate the natural environment. Multiple air pumps were also placed close to the aquarium walls to provide ample oxygenation and additional regions with turbulent flow. To promote algal growth, an over-tank light-emitting diode (LED) light was set to a 12-hr day-night cycle. The aquarium was kept in a 4°C climate room (mean temperature of 3.2°C ± 0.9°C; mean ± SD) to replicate alpine stream temperatures.

SEM was used to image fourth instar H. lugubris larvae as described previously (Kang et al., 2019). In brief, samples fixed in 70% ethanol (v/v) were flash-frozen in liquid ethane cooled with liquid nitrogen and freeze-fractured immediately afterwards with a double-edged razor blade on a cooled aluminium block to obtain longitudinal views. Samples were freeze-dried overnight, then carefully mounted on SEM aluminium stubs using carbon tape and silver paint. They were then sputter-coated with 15 nm of iridium and imaged using a field-emission SEM (FEI Verios 460).

One H. lugubris fourth instar larva was fixed in 2% paraformaldehyde and 2% glutaraldehyde (v/v) in 0.05 M sodium cacodylate buffer (pH 7.4) for 7 days at 4°C. The larva was then dissected into six pieces—each containing one suction organ—and fixed for an additional day. The samples were then rinsed multiple times in 0.05 M sodium cacodylate buffer followed by deionised water before dehydration through a graded ethanol series: 50%, 75%, 95%, 100% (v/v), and 100% dry ethanol. The dehydrated samples were critical-point dried using four flushes of liquid CO₂ in a Quorum E3100.

One critical-point-dried suction organ was used for imaging via micro-CT. The sample was mounted on a standard dressmaker’s pin using ultraviolet (UV)-curable glue, then imaged using a lab-based Zeiss Xradia Versa 520 (Carl Zeiss XRM, Pleasanton, CA, USA) x-ray microscope. The sample was scanned at 0.325 µm/pixel with an accelerating X-ray tube voltage of 50 kV and a tube current of 90 µA. A total of 2401 projections collected at 20 s exposure intervals were used to perform reconstruction using a Zeiss commercial software package (XMReconstructor, Carl Zeiss), utilising a cone-beam reconstruction algorithm based on filtered back-projection. Subsequent 3D volume rendering and segmentations were carried out using Dragonfly v4.0 (Object Research Systems Inc, Montreal, Canada) and Drishti v2.6.5 and v2.7 (Limaye and Stock, 2012).

Insect attachment forces were measured using a custom centrifuge set up described previously (Federle et al., 2000). The centrifuge operated on the following principle: a platform with the test substrates and the insect was driven by a brushless motor, and a light barrier sensor was triggered per rotation. This signal was used to synchronise image acquisition from a USB camera (DMK 23UP1300; The Imaging Source Europe GmbH, Bremen, Germany), and image frames and their corresponding times were recorded using the StreamPix4 software (NorPix Inc, Montreal, Canada). For safety reasons, the maximum centrifugation speed was limited to approximately 75 rotations per second (rps). Some of the blepharicerid larvae could not be detached even at the maximum speed (n = 14 out of 136 measurements); in such cases, we used the maximum acceleration of a successfully detached individual from the given species.

Effect of surface roughness on the peak shear force of blepharicerid larvae was measured on the following substrates: smooth (clean polyester films), micro-rough (polishing films with a nominal asperity size of 0.05 µm; Ultra Tec, CA, US), and coarse-rough (30 µm polishing films; Ultra Tec). The same substrate types were used to measure the normal force (substrates mounted vertically in the centrifuge), but a polished polymethyl methacrylate (PMMA) surface was used as the smooth substrate. Surface characteristics (average roughness (mean height deviation) R_a, root-mean-squared roughness R_q, and maximum peak-to-valley height (PV)) of the micro-rough substrates were obtained using white-light interferometry with a scan area of 0.14 × 0.10 mm (Zygo NewView 200; Zygo Corporation, CT, USA). Micro-rough substrates were sputter-coated with 5 nm of iridium prior to scanning to improve the surface reflectivity. As the coarse-rough substrate could not be adequately imaged via white-light interferometry, we used a Z-stack image focal-depth analysis technique as described elsewhere (Sarmiento-Ponce et al., 2018) with a scan area of 0.44 × 0.58 mm. For both surfaces, three regions were selected at random and imaged. Interferometry images were analysed using MetroPro software (Zygo), and a custom MATLAB script was used to reconstruct the surface profile from the Z-stack images (The MathWorks Inc, MA, United States).

Since L. cinerascens and L. cordata were difficult to maintain in laboratory conditions, these two species were tested only on smooth horizontal surfaces (n = 43 and n = 10, respectively). The full range of tests on smooth, micro-rough, and coarse-rough surfaces was conducted for H. lugubris (n = 9–15 for shear tests; n = 10 for all tests in normal direction). Prior to the experiments, individuals were selected from the laboratory aquarium and placed inside specially prepared 50 ml Falcon tubes. This tube was kept on ice for the duration of the experiment. For each run, a larva was carefully removed from the tube and placed on the test surface. A droplet of water (taken from the aquarium) was used to wash excess debris from the insect, and lab tissue paper was used to wick away excess water without removing all moisture from the larva; the contact zone of the suction discs was still completely wetted under these conditions, as confirmed by IRM observations. These steps were necessary to prime the larvae for the centrifugation trials as they often displayed defensive behaviour while being handled. The larvae adhered and remained still once primed, and between two and four repetitions were performed for each larva. All centrifuge trials were conducted within 7 days of collection.

After the trials, all the larvae were blot-dried on filter paper and weighed using an analytical balance (1712 MP8; Sartorius GmbH, Göttingen, Germany). Statistical analyses were conducted on log₁₀-transformed values using R v3.6.2 run in RStudio v 1.2.5033 (R Development Core Team, 2019; Team RS, 2019).

Normal (adhesive) stress and shear stress (defined as the peak attachment force divided by the contact area) were calculated using suction disc areas measured for L. cordata and H. lugubris larvae. Larvae were placed on microscope slides so that the suction organs fully contacted the glass and imaged with a stereomicroscope. Every tested L. cordata and H. lugubris specimen was imaged. A representative organ was selected from each L. cordata and H. lugubris larva, and the contact area calculated by fitting a circle inclusive of the outer fringe layer using FIJI (Schindelin et al., 2012) (https://imagej.net/Fiji). The peak attachment force was then divided by this contact area to determine the peak stress for each larva.

C. morosus (Sinéty, 1901) stick insects were used as a model for terrestrial insect adhesion, and their attachment on surfaces with varying roughness was measured to compare against blepharicerid larval attachment. Second instar nymphs with undamaged legs and tarsi were selected for centrifuge experiments using smooth, micro-rough, and coarse-rough surfaces (n = 10 per surface). No normal forces could be measured on the micro-rough surface as stick insects failed to hold their body weight during preliminary tests. Before each trial, we checked that the specimen was in contact with the surface using all six legs and that the surface was uncontaminated. Stick insects were oriented with the head facing out, and each individual was tested twice and weighed afterwards. The higher attachment force per individual was used as the peak attachment force.

In order to examine blepharicerid larvae locomoting for extended periods of time, a custom flow chamber was built to imitate the fast-flow conditions of their natural environments (Figure 8). Two aluminium plates (approximately 60 × 100 mm in height × width), each with a rectangular window, were used to sandwich an inner chamber made out of polydimethylsiloxane (PDMS; Sylgard 184, Dow Corning, MI, USA). This inner chamber had a lemon-shaped chamber to serve as the observation arena, and an inlet and an outlet for water circulation. Two microscope coverslips (0.16–0.19 mm thickness; Agar Scientific, Stansted, UK) were used to encase the inner chamber. Two to five larvae were placed on the bottom coverslip, and once the top coverslip was placed over the arena, four clamps were used to squeeze the aluminium plates and coverslips against the PDMS. The soft PDMS moulded closely to the plates and created a water-tight seal. Aquarium water (kept cool in an ice bath) was pumped via a micro-pump (M200S-V; TCS Micropumps Ltd, UK), and the input voltage was controlled by a microprocessor. The flow rate was controlled by setting an appropriate pump-operating voltage. With this flow chamber, we recorded H. lugubris larvae locomotion and the attachment/detachment of suction organs on smooth glass surfaces via IRM, which has been previously used to investigate the contact between animal adhesive organs and the substrate (Federle et al., 2006; Federle et al., 2002). Videos were recorded using a USB camera (DMK 23UP1300) and the IC Capture software (v2.4.642.2631; The Imaging Source GmbH) at 30 frames per second (FPS).

Figure 8

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To observe how suction organs respond to surface roughness, we used transparent micro-structured surfaces with well-defined micro-ridges and grooves fabricated by photolithography and nanoimprinting (Zhou et al., 2014). In brief, a master surface was first produced using photolithography, and a PDMS mould of this master was used to cast the final surface out of epoxy. Three micro-ridge geometries were used in our experiments: (1) 3 × 3 × 2 µm (ridge width × groove width × ridge height); (2) 3 × 3 × 4 µm; and (3) 10 × 10 × 2 µm. As the ridge height is only approximately controlled through the spin-coating of photoresist when producing the master, we measured it from the epoxy replicas using white-light interferometry as mentioned above (see Table 1). Four to five regions from each uncoated substrate were imaged and only regions without artefacts were used in the final calculation. For simplicity, when referring to the substrates, the depths of the grooves were reported to one significant figure (ie, 3 × 3 × 2 µm, 3 × 3 × 4 µm, and 10 × 10 × 2 µm, for widths of ridges, grooves, and ridge height). Note that as these surfaces could not be used in combination with the flow chamber, a H. lugubris larva was placed on the substrate, wetted with a droplet of aquarium water, gently motivated with soft-touch forceps, and recorded as they moved around on the substrate. One H. lugubris specimen was used for both the 3 × 3 × 2 µm and 10 × 10 × 2 µm substrates, and a different larva was used for the 3 × 3 × 4 µm surface.

Based on our observation that the microtrichia never showed any side contact, even when the suction discs were in contact with micro-structured substrates, we estimated the minimum elastic modulus of the microtrichia cuticle. We estimated the maximum force F on one microtrichium, perpendicular to the surface, as 56 nN. This was obtained based on the following assumptions: (1) the suction discs are loaded with a peak normal force of 11.6 mN, ie, 1.9 mN per sucker (from centrifuge measurements of H. lugubris); (2) equal loading of ca 34,000 spine-like microtrichia in tip contact (based on the area of the suction disc bearing spine-like microtrichia of ca 49,000 µm², with the average microtrichia tip density of 0.7 per µm²). The microtrichia were assumed to be cylindrical, with a length $L=6.7\pm 0.5\mu m$ and diameter $D=0.56\pm 0.01\mu m$ (mean of means ± standard error of the mean; n = 2 H. lugubris), and the angle between the unloaded microtrichia and the surface was estimated as $\alpha =45\pm 3°$ (mean ± SD measured from five microtrichia of H. lugubris). The local adhesion and friction force of the microtrichium were assumed to be negligible.

Following Goss and Chaouki, 2016, the elastic modulus below which a cylindrical beam loaded at an angle α (see ${\varphi }_a$ definition) would exhibit side contact is

where $K\left(p^2\right)$ and $F\left({\varphi }_a|p^2\right)$ are the complete and incomplete elliptic integrals of the first kind, respectively, $p^2=1/2$ is the elliptic modulus, ${\varphi }_a={sin}^{-1}\left(\frac{sin\alpha /2}{p}\right)$ is the elliptic amplitude, and $I=D^4\pi /64$ is the second moment of area. See Figure 6d–ii for a schematic of a hypothetical scenario where microtrichia make side contacts on a smooth surface.

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

We would like to thank P Ladurner for assisting in sample collection and providing laboratory space near to the field site and M Sutcliffe for helping with the surface profilometry measurements. We are also grateful to KH Muller and JN Skepper at the Cambridge Advanced Imaging Centre for their help in preparing and imaging SEM samples.

1. Received: September 18, 2020
2. Preprint posted: October 2, 2020 (view preprint)
3. Accepted: September 24, 2021
4. Version of Record published: November 3, 2021 (version 1)

© 2021, Kang et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.