Framework with cytoskeletal actin filaments forming insect footpad hairs inspires biomimetic adhesive device design

Insects capable of walking on smooth surfaces use specialized adhesive organs, footpads, composed of flexible setae whose tips exhibit spatulate, discoidal, or pointed microstructures. Adhesion is mediated by Van der Waals, Coulomb, and capillary forces generated by a lipid-like, non-volatile secretion bridging the setal tip and substrate. While footpad morphology and function are relatively well characterized, the developmental mechanisms that generate these microstructures remain poorly understood. Drosophila melanogaster provides a powerful genetic model to study footpad development; its pretarsi bear rows of spatula-tipped hairs. Prior work implicated Polycomb group gene Su(z)2 in adhesive pad differentiation and climbing ability, but detailed molecular and cellular mechanisms have not been established. This study investigates how cytoskeletal actin shapes footpad hair tips during metamorphosis and how such principles can inspire a biomimetic adhesive device.

Classical and modern studies have described insect tarsal attachment structures, detailing setal tip shapes across taxa and the roles of secreted fluids in adhesion via capillary forces and intermolecular interactions. Chemical analyses identified lipid-like components in footprints of ladybird beetles. Mechanical studies measured adhesion forces at setal terminal plates and highlighted direction-dependent friction/adhesion differences between smooth and hairy pads. In Drosophila, adhesive pads with spatulate hairs have been anatomically characterized, and a Su(z)2 mutant exhibited malformed pads and impaired climbing. Material gradients in insect setae (e.g., ladybird beetle) suggest functional stiffness tuning along hair length. Biomimetic research since 2005 largely focused on dry adhesives inspired by geckos; however, on rough surfaces, maintaining real contact area is challenging without compliant tips and fluid-mediated adhesion. These findings collectively motivate probing cytoskeletal contributions to setal morphogenesis and leveraging wet adhesion mechanisms for device design.

Model organism and staging: Drosophila melanogaster (Canton-Special) were reared on cornmeal-yeast medium at 25 °C under constant light. White prepupae were collected within 30 min after puparium formation (APF) and aged at 25 °C. Labeling footpad cells: Multiple GAL4 drivers involved in pretarsus development (bar, ckm10, spa, neur, dpp, svp) were crossed to UAS-mCD8::GFP to assess expression around ~48 h APF; svp-GAL4 lines robustly labeled footpad hair cells and were used for experiments. Hair cells were visualized using svp>Act5C::GFP and nuclear DsRed (svp>Act5C::GFP, nucDsRed). Confocal imaging tracked morphology from 20–40 h APF. MARCM single-cell labeling: To determine cell-to-hair mapping, somatic clones were generated (y hs-flp; FRTG13 UAS-mCD8::GFP/FRTG13 TubP-GAL80; svp-GAL4/+). Heat shock at 37 °C for 30 min produced sparse labeling; preparations were fixed and immunostained. Actin visualization: Fixed pupal pretarsi were stained with fluorescent phalloidin (TexasRed-X phalloidin, Acti-stain 555, Alexa Fluor 633) to localize F-actin over developmental timepoints (24, 30, 40 h APF). Anti-GFP immunostaining was used when appropriate. Imaging used a Leica TCS SPE confocal microscope. Ultrastructure and morphology: Adult footpad morphology was examined by SEM. Legs were fixed (3.7% formaldehyde), dehydrated through ethanol, tert-butanol exchanged, freeze-dried, gold-coated, and imaged (TM3000). TEM of hair cross-sections used glutaraldehyde/paraformaldehyde fixation, OsO4 postfixation, resin embedding, ultrathin sectioning, uranyl acetate and lead staining, and imaging on JEOL JEM-1220. RNAi-mediated Actin5C knockdown: UAS-Act5C RNAi was expressed in footpad hair cells with svp-GAL4. To mitigate lethality, two strategies were used: (1) temporally restrict RNAi post-pupariation with TubP-GAL80TS (larvae at 18 °C; 0–12 h APF shifted to 30 °C; genotype svp>Act5C RNAi, TubP-GAL80TS), and (2) suppress GAL4 activity in neurons with elav-GAL80 (svp>Act5C RNAi, elav-GAL80). Adult survivors were analyzed by SEM for tip morphology. Malformed (forked) tips were quantified among 15 hairs from three distal rows per stalk per footpad. Live imaging: For time-lapse imaging (every 20 min), pupae with svp>mCD8::GFP, nucDsRed or svp>Act5C::GFP, nucDsRed were mounted facing down on a coverslip over a humid chamber to prevent desiccation and imaged through to framework completion. Behavioral assay (climbing): Individual females (24–48 h post-eclosion) were placed in glass Pasteur pipettes sealed with cotton. After tapping to the bottom, climbing to the constriction point (8 cm) was recorded for 1 min. Outcomes: time to reach constriction (excluding those failing within 1 min for mean calculation) and percentage overcoming the constriction within 1 min. Genotypes: +/+ (CS), +/+; svp-GAL4/+, UAS-Act5C RNAi/+; elav-GAL80/+ (controls) and UAS-Act5C RNAi/+; svp-GAL4/elav-GAL80 (knockdown). Biomimetic device fabrication: Two nylon fibers (52 µm diameter) spaced 2 mm apart were fixed at one end to paper. Fibers were dipped into 1 wt% sodium alginate solution and withdrawn; surface tension formed a spatulate film between fibers. The assembly was immersed in 1 wt% calcium lactate to gel the alginate (calcium alginate). After air-drying, spatulae were cut from the paper. Before testing, structures were hydrated. The spatulate area was 1.2 ± 0.1 mm² (mean ± SE, n=11). All steps at 23 °C. Adhesion testing (shear): Glass substrates were ultrasonically cleaned (acetone, ethanol, water; 10 min each). Devices were dipped in water (loaded water mass 560 ± 5 µg, mean ± SE, n=6) and tested at 23 °C using a 980 mN load cell. Shear tests translated the substrate laterally at 24 mm/min. Statistics and reproducibility: One-way ANOVA with Tukey–Kramer post hoc tests assessed malformed hair percentages and climbing times. The proportion overcoming the constriction used Kruskal–Wallis with Scheffé post hoc tests. Sample sizes and P-values are reported in figures; qualitative imaging was replicated at least five times with consistent results.

- Developmental morphogenesis: In Drosophila pretarsi, ~30 spatula-tipped hairs are arranged in 6–7 rows per footpad. TEM showed each hair is a flat tube composed of cuticle. During pupal development, svp-expressing hair cells extend processes while cell bodies shift proximally; by ~40 h APF, F-actin assembles at hair tips into a spatulate framework, followed by cuticle deposition. MARCM demonstrated a one-to-one mapping: a single cell forms a single hair. - Actin requirement: Phalloidin staining revealed apical F-actin accumulation at 24 h APF, extension of actin-rich processes by 30 h APF, and a spatulate actin framework at 40 h APF. RNAi knockdown of Actin5C in hair cells (with either TubP-GAL80TS timing or elav-GAL80 neuronal suppression) produced forked, malformed hair tips instead of normal spatulae. Other gross aspects of footpad formation appeared unaffected under the knockdown levels used. - Functional impact on adhesion/locomotion: Act5C knockdown flies exhibited impaired climbing on smooth glass in a Pasteur pipette: approximately twice the time to reach the constriction compared with controls, frequent slipping, and significantly reduced success in overcoming the constriction (multiple comparisons with very low P-values, e.g., P ~10^−11 to 10^−28). These deficits are consistent with reduced effective contact area due to malformed tips. - Biomimetic device inspired by development: Emulating the two-step biological process (framework then deposition), a fiber-framed adhesive structure was fabricated using two nylon fibers and a calcium alginate spatula formed by surface tension and gelation. Adhesion depended strongly on water content. Immediately after dipping (higher water content), shear adhesion was 40 ± 15 mN (mean ± SE, n=6). When nearly dry after contact, shear adhesion increased to 779 ± 108 mN (mean ± SE, n=11). The corresponding shear stress was 784 ± 144 kPa, comparable to distal beetle pad shear stress on smooth surfaces (≈613–642 kPa). A single fiber could suspend a 52.8-g silicon wafer, and the device could suspend an estimated 60-kg load with an adhesive area of ~9 cm² (≈756 fibers) under optimal conditions. Adhesion was reversible and repeatable; it decreased with increased liquid volume and increased as the liquid diminished, consistent with transitions from capillary forces to Laplace pressure and then intermolecular forces as gap thickness decreases. - Scaling: The artificial spatula’s attached area (1.2 ± 0.1 mm²) is about three orders of magnitude larger than a fly hair tip (~3 ± 0.1 µm²), yet both operate with film-like geometries where surface effects dominate, highlighting the generality of elastocapillary mechanisms across scales.

The study identifies a cytoskeletal mechanism for shaping adhesive microstructures in insect footpads: hair cells elongate and assemble F-actin bundles into a spatulate framework that templates subsequent cuticle deposition. Disrupting actin (Act5C knockdown) compromises tip morphology and reduces adhesive performance during climbing, directly linking actin-mediated morphogenesis to function. The conserved, cell-autonomous process (single cell forms single hair) suggests an evolutionary module for generating diverse setal tip shapes across insects via modulation of actin framework geometry. Translating this developmental principle to engineering, the authors implemented a two-step fabrication mimicking framework formation and material deposition using nylon fibers and alginate gelation driven by surface tension. The resulting device exhibits high shear adhesion on smooth glass, reversibility, and sensitivity to liquid amount akin to biological pads. The compliance and hydration at the tip increase real contact area and adhesion on roughness, while the fiber frame provides tensile strength and durability. The device’s shear stress matches that of insect distal pads, supporting the effectiveness of developmental biomimetics. The findings emphasize the roles of structural compliance, interfacial liquid volume, and elastocapillary interactions in adhesion, offering design rules for scalable wet-adhesive systems.

By elucidating that F-actin frameworks sculpt spatulate footpad hair tips in Drosophila and are essential for effective adhesion, this work bridges developmental cell biology with bioinspired materials engineering. The biomimetic fiber-framed alginate spatula, fabricated via a simple self-assembly process, achieves high, reversible shear adhesion comparable to insect pads and functions repeatedly with minimal energy and low-cost materials. This demonstrates developmental biomimetics as a viable route for designing novel adhesion devices. Future work should quantify structural compliance, optimize resistance to normal (peel) loads, assess performance across substrates and roughness scales, refine liquid management for tunable adhesion, and explore miniaturization and arrayed architectures for practical applications.

- Genetic manipulation: Act5C knockdown levels were tuned to avoid lethality and may not reveal all roles of actin; only tip malformations were evident under these conditions. - Behavioral assays: Climbing performance was assessed on smooth glass; adhesion on varied substrates or roughness scales was not quantified for flies. - Device performance: Adhesion was primarily characterized in shear on smooth glass; the device is weak against normal (peel) loads and requires rehydration (dipping in water) when fully dried. Compliance of the device was not quantified, and comparisons to insect pads would benefit from detailed mechanical compliance measurements. The artificial contact area is orders of magnitude larger than biological hairs, so direct scaling to microscopic arrays remains to be demonstrated.