Protopodia protrusions in each segment are sequestered during swing phases of forwards locomotion.

a, Schematic of setup for lateral imaging of larvae, using confinement in Pasteur pipette pre-filled with 0.1% (w/v) agarose. To encourage forward crawling, 10μl of 15mM ethyl butanoate (EB) was placed as attractive odour at the end of the pipette. b, Lateral brightfield image of 3rd instar larva showing convex areas of denticle bands (open arrowheads) protruding into the substrate, interdigitated by concave areas of naked cuticle (black line) not interacting with the substrate. Scale bar=750μm. c, Time lapse of area marked by dotted box in b showing the swing periods and stance periods of protopodia (coloured open arrowheads and dotted lines) during a forward wave. Red and blue dots at 0s denote anterior and posterior rows of denticles, respectively. As the posterior-most denticle row moved to meet the anterior row of the band, the medial row detached from the substrate via invagination (white arrows). The invaginated pocket is then moved forward (black arrow) and subsequently replanted. This action repeats as the wave propagates. Scale bar=500μm. Images representative of three 3rd instar larvae.

Protopodia kinematics follow ‘heel-toe’-like footfall dynamics.

a, (i) Brightfield image and (ii) schematic of 2nd instar larvae showing ventral side denticle belts which reside upon the protopodia and (iii) schematic of the imaging setup used for kinematic tracking. Scale bar=200μm. b, (i) As a forward wave travels through the animal, the distance between denticle bands decreases. Scale bar=200μm. (ii) At higher frame rate and magnification, changes in distance between the posterior and anterior most denticle rows are resolved. The posterior-most row (P, blue) initiates movement first and moves until nearly reaching the anterior-most row (A, red) at 0.544s, after which point, they move together (0.561s). Scale bar=100μm. c, Velocity of anterior- and posterior-most denticles rows (A2d A/P, A4d A/P, A6d A/P) and the left/right end of denticle bands (A2 L/R, A4 L/R, A6 L/R and A8 L/R) over three representative forward waves, showing how the strategy observed in b is maintained across body segments. Background colours indicate swing initiation (SI, blue), swing period (SwP, light grey), swing termination (ST, pink) and stance period (StP, dark grey). d, Forward wave latency for different animals and body segments. Positive values denote posterior row led latency. n=10 animals, 30 waves. e, SI-latency is correlated with wave duration in the posterior abdomen (A6: R2=0.61, purple; A4: R2=0.78, red) but less so for the anterior abdomen (A2: R2=0.35, yellow). n=12 animals with 3 latency periods per segment. f, ST-latencies show no correlation to wave duration (A6: R2=0.26, A4: R2=0.26, A2: R2=0.03). n=12 animals with 3 latency periods per segment.

ERISM maps mechanical substrate interactions in Drosophila larvae.

a, Schematic of setup for ERISM with Drosophila larva on an optical microcavity. Maps of local cavity deformation (displacement) due to indentation forces are generated by analysing cavity resonances. b, Force distance relationship measured by AFM and c, Mechanical stiffnesses (Young’s moduli) for microcavities produced by mixing different elastomers at different ratios and applying different plasma conditions. d, g, j, Brightfield images of anaesthetised 2nd instar larvae recorded at low, medium, and high magnification. e, h, k, Corresponding maps of microcavity displacement. (* denotes contamination on cavity surface from handling the larva.) f, i, l, Corresponding maps of mechanical stress obtained by finite element analysis of displacement maps, showing the stress on the substrate due to passive interaction between larvae and substrate. Scale bar=500μm (d), 250μm (g) and 50μm (j). Images representative of 4 separate 2nd instar larvae. Microcavities in d-i used 30W O2 10% Sylgard®184 design, and j-l used a 30W O2 5% Sylgard®184 design.

WARP imaging reveals dynamics of substrate interactions during larval movement.

a, WARP image sequence of displacement and stress maps (top) for a freely behaving 2nd instar larva during forward locomotion. (*denotes dust artefact.) Lateral projections of stress maps (bottom) showing individual protopodia interdigitated by naked cuticle. As a contractile wave (grey box) progressed through the animal, protopodia were lifted off the substrate. Scale bar=100μm. b, WARP image sequence of larva prior to (−1.5s to -0.5s) and engaging in (0s) a headsweep (representative of 2 animals and 3 turns). Note the large posterior displacement (blue arrow). (Images cropped around the animal.) Scale bar=200μm. c, Profiles of cavity displacement along anteroposterior (A-P) axis in resting state (black dotted line at -1.5s in b) and pre-headsweep (red dotted line at -0.5s in b), showing that peak displacement decreased across all segments from the resting state (grey box) to pre-headsweep (pink box). d, Bilateral displacement profile across the mediolateral (ML) axis of the A4 protopodium (solid lines in b) at different times prior to the headsweep, showing that the width of the contact increases from the resting state (−1.5s) to the pre-headsweep state (−0.5s) and partially reduces again immediately after head movement. e, (i) Brightfield image (3rd instar larva) and (ii) displacement map (2nd instar larva) of the posterior-most body segment, showing how two cuticular protrusions (white arrowheads) and the terminal protopodium (A8) generate a tripod-shaped substrate displacement. (iii) Profiles along blue and red dotted lines in (ii). Scale bar=200μm (i) and 100μm (ii). f, Sequence of displacement maps of tripod structure before the start of a forward wave (<0.24s) and the removal of tripods upon beginning of peristalsis (>0.48s). Scale bar=100μm. g, Percentage of forward waves (FW), bilateralisms (BL), backward waves (BW) preceded by tripod contact, and tripod deployments without any observed locomotor behaviour (unrelated). h, Time delay between tripod deployment and initiation of movement at A7. Points colour-coded by animal, n=6. Line=mean, box=±1 standard error of the mean, whiskers=±1 standard deviation.

Protopodia produce GRFs in the micronewton range and show complex spatiotemporal dynamics.

a, Ground reaction force (GRF, coloured line) and protopodial contact areas (white area under black line) during forward crawling for A2, A4 and A6 protopodia, showing progression of waves through animal (light-coloured boxes). Blue (SI) and pink (ST) boxes denote characteristic troughs in GRF immediately prior to protopodia leaving the substrate and returning to the substrate, respectively. b, Volume displaced by different protopodia show a 2nd order polynomial relationship with the contact area of that protopodium (A6: R2=0.91, A4: R2=0.91, A2: R2=0.88). c, Peak GRFs and d, peak contact area during SI and ST across body segments. Data points denote single events, colours indicate different animals. n=5, 15 waves. Contact areas were compared by a two-way repeated measures ANOVA (*<0.05, **<0.005, ***<0.0005, n.s.=not significant). e, During SI, peak displaced volume was correlated to wave duration for larger abdominal segments (A6: R2=0.69; A4: R2=0.48) but not for smaller anterior segments (A2: R2=0.24). During ST, it was not correlated regardless of the segment (A6: R2=0.05; A4: R2=0.05; A2: R2=0.08). n=4, 11 waves.

Sub-protopodial force dynamics reveal sub-step processes and functional substrate interfacing domains in each step.

a, WARP imaging of protopodial landing during ST of an A6 protopodium. Raw interference images from WARP acquisition show footprints of individual denticles as white dots. Displacement and stress maps show how landing starts with posterior denticle rows before spreading out along the AP and ML axes. Scale bar=100μm. b, (i) Displacement map of whole animal. (ii) Kymograph of displacement along AP axis (black line in i) over 2 forward waves. Bands of red and blue correspond to naked cuticle and protopodia, respectively. Scale bar=100μm. c, (i) Kymograph of displacement along the AP axis of an A6 protopodium (box in b). (ii) Profiles across kymograph at different positions along the AP axis of protopodium (lines in i). (iii) Latency of substrate indentation (displacement <0nm) during ST along the AP axis, relative to the extreme posterior of protopodium. Compared to the posterior half of protopodium (light blue area), the anterior half shows larger latencies and variations in latency (light red area). n=4 animals, 8 swing termination events. d, Kymograph of displacement along AP axis during ST for the distal left (dL), medial left (mL), midline (m), medial right (mR) and distal right (dR) section of the A6 protopodium. Height of each kymograph, 66.42μm, (ii) Profiles across the central AP line of each kymograph in (i). Vertical lines indicate times when midline, medial right/left and distal right/left indentation starts (displacement <0nm). (iii) Latency of substrate indentation during ST relative to the midline for medial right/left and distal right/left locations. n=4, 8 swing termination events.

Proposed model for protopodia-substrate interactions during Drosophila larval locomotion.

a, Schematic illustration of forward wave propagating from posterior (blue) to anterior (yellow). b, At the start of a forward wave, animals contract the posterior-most abdominal segment (A8), producing an anterograde horizontal force Fh(A8). Due to Newton’s 3rd law, there is an equal but opposite reaction force -Fh(A8). To counteract this force, tripod processes (TPs) deploy onto the substrate and generate a temporary anchor, allowing the A8 protopodium to swing forward. c, During swing termination (ST) at the end of the swing period (SwP) of segment An, the corresponding sequestered protopodium (Sq. n) strikes the substrate with its posterior most denticle row, then gradually unfolds into the substrate along its entire anteroposterior extent. During the stance period (StP), this planted segment n (Ptd. n) forms an anchor to mitigate the retrograde reaction force due to the subsequent contraction of segment n-1. d, In time with anchoring of protopodium n, protopodium n-1 performs swing initiation (SI) by removing denticles from the substrate and sequestering into an invagination pocket, which reduces friction during the subsequent SwP. The contraction of segment n-1 then leads to an anterograde force (Fh) that is balanced by the anchoring of protopodium n as illustrated in c.