SEM and CLSM imaging of tsetse tarsi.

A-C: Ventral views of the tarsi of the first (A, B), and second (C) leg pairs. D-E: Magnified views of the pulvillus from C. F: Lateral overview of the pulvillus and the ungues. G-H: Details of the pulvillus from F. Shown are the spatulas from a ventrolateral view at the rear of the pulvillus with signs of deformation (G), and further apart from the margin with no deformation (H). I: CLSM imaging representing compositional differences in the insect cuticle: flexible, resilin-rich regions appear blue, moderately stiff regions range from green to yellow, and rigid, highly sclerotized zones show up in red. Yellow planes on the cubes indicate the orientation of the tarsus attachment surfaces. Abr.: ac, acanthae; cr, crest; fr, furrow; j, joint; ot, oblate tip; pt, pretarsus; pvl, pulvillus; set, setae; sr, surface ridge; tm, tarsomeres; ung, ungues.

Blood-feeding tsetse fly and SEM images of tsetse tarsal adhesive structures.

A: A tsetse fly taking a blood meal on a silicone feeding mat. B: Ventro-lateral overview of the pulvillus with a magnified view of the acanthae, showing fine crests on the underside and surface modifications (green) on the upper side. The yellow plane of the cube indicates the orientation of the tarsus attachment surface. Abr.: ac, acanthae; cr, crest; fe, femur; fr, furrow; ot, oblate tip; pr, proboscis; pvl, pulvillus; spa, spatula; ti, tibia; tm, tarsomeres.

Tarsal attachment performance of tsetse flies expressed in friction force and safety factor.

A: Schematic of the experimental setup for attachment measurements. The rotational plate, powered by a motor, accelerates until the fly loses grip, which is detected by a light sensor. B-C: Boxplots of friction forces at which male (B) and female (C) flies detached from different surfaces. Ten flies per sex were tested and for each surface, 3–14 replicates were averaged per fly. Graphs display these per-fly means, with boxes spanning the interquartile range, whiskers the full range, and a line marking the median. D: Illustration of how the acanthae interact with surfaces of different topographies. E-F: Safety factors for male (E) and female (F) flies on different surfaces. The safety factor, defined as total friction force divided by body weight (i.e., g-force), normalizes fly weight for cross-individual comparison. It was calculated for each replicate in B–C, and per-fly means were plotted. Boxplots are constructed as above. G-H: Boxplots of the friction forces (G) and safety factors (H) across all substrates, compared between sexes, based on all per-fly means. Boxplots are constructed as above. Statistical significance in B, C, E, and D was determined using the Friedman test; *, p<0.05; **, p<0.001; ***, p<0.001; ****, p<0.0001. Comparisons between male and female flies in G and H were performed using the Mann-Whitney test, revealing no significant differences. All measured values, friction forces, and safety factors are provided in Supplementary material 1. Abr.: co, cover; gl, glass; er, epoxy resin (smooth); ls, light sensor; ps, surface plate; ts, tsetse fly.

3D-reconstruction of the tsetse head.

A: Frontal view rendering of a tsetse head derived from micro-computed tomography (µCT) data. Video 2 provided with this manuscript shows a 360° rotation of this 3D rendering. B-C: Frontal views of the 3D-reconstructed model from the same dataset, showing the external surface (B) and internal structures (C) D: Lateral view of C. E: Enlarged view of D emphasizing muscular structures associated with the proboscis and suction pump. Abr.: ant, antennas; at, alimentary tract; b, brain; c, caput; ce, compound eye; dil, pharyngeal dilator muscles; hy, hypopharynx; lb, labium; lr, labrum; me, median eye; ml, labial muscles; mx, maxillary palp; p, proboscis; ol, optical lobe; sd, salivary duct; sp, suction pump; tr, trachea; nt, thoracic nerve.

Rendering of a tsetse head derived from micro-computed tomography (µCT) data.

Related to Figure 3. A: Frontal view. Video 2 provided with this manuscript shows a 360° rotation of this 3D rendering. B: Lateral view. C: Lateral view of the digitally generated cross section. Abr.: ant, antenna; b, brain; ce, compound eye; dil, pharyngeal dilator muscles; lb, labium; lr, labrum; lnm, longitudinal muscles; me, median eye; ml; labial muscles; mx, maxillary palp; pr, proboscis; sp, suction pump; tho, thorax.

SEM micrographs of the tsetse proboscis and labellum joint details.

A: Frontal view of the head showing the exposed ventral mouthparts. B-C: Enlarged views of A, focusing on the bulbus (B) and the closed labellum (C). D: Dorsal overview of the mouthparts. E-F: Magnifications of D, showing the dorsal base of the proboscis and maxillary palps (E), and the dorsal side of the opened labellum (F). G-I: Successive magnifications of a tsetse head after removal of the proboscis. The labrum displays hollow segments along its lateral walls (I). J-L: Views of a dissected proboscis. The schematic to the right indicates the separation site in blue, and the blue areas in the SEM images mark the corresponding cut surfaces where the parts were previously connected. Shown are the proximal portion (i.e. base to mid-length) of the proboscis as dorso-frontal overview (J) and a magnified detail of this highlighting the furca and labial gutter (K), as well as a view into the distal tip of the labellum (L). Abr.: bu, bulbus; ce, compound eyes; dt, dorsal teeth; f, furca; gs, gustatory sensilla; hy, hypopharynx; lb, labium; lg, labial gutter; lm, labellum; lr, labrum; mx, maxillary palps; rsp, rasping teeth; tn, tendons.

FIB-SEM imaging of trypanosome-infected and uninfected tsetse labella and SEM imaging of the dissected proboscis.

Related to Figure 5. A: The labellum was imaged gradually layer by layer from the tip using FIB-SEM, reconstructed in 3D, and a ventral view of the interior was generated, as indicated by the purple plane. B: Ventral overview of the inside of the digitally dissected closed labellum. C: Magnified view of B, trypanosomes are highlighted in orange. D: Lateral overview of a dissected head and proboscis, exposing labial muscles in the bulbus. E: Close-up of C, showing parts of the dissected labium. F: Magnified and rotated view of D, showing the labial gutter and part of the hypopharynx. Abr.: ce, compound eye; hy, hypopharynx; lb, labium; ml, labial muscles; pt, prestomal tooth; rsp, rasping teeth; sc, scales; tryp, trypanosomes.

FIB-SEM and SEM imaging of the tsetse labellum and associated tooth structures.

A: 3D reconstruction from FIB-SEM data showing the internal structure of the closed labellum in frontal view. The orange arrow indicates trypanosomes. A ventral view of the closed labellum is shown at the side for orientation. Video 3 provided with this manuscript shows a sequential progression through the labellum based on the FIB-SEM dataset. B-G: SEM micrographs of labella in progressively opened states; scale bars = 100 µm. H: Ventral overview of the proboscis with an open labellum. I: Close-up from H, highlighting exposed teeth-like structures. J: Enlarged view of I with focus on the rasping teeth. Abr.: bu, bulbus; dn, denticles; dt, dorsal teeth; fs, flagelliform sensilla; gs, gustatory sensilla; hy, hypopharynx; lg, labial gutter; mx, maxillary palps; pr, proboscis; pt, prestomal tooth; rs, rasping teeth; sc, scales; sl, small sensilla; vt, ventral teeth.

CLSM maximum intensity projections of tsetse probosces in different opening states, highlighting resilin distribution and sclerotization.

The colour gradient represents compositional differences in the insect cuticle: flexible, resilin-rich regions appear blue, moderately stiff regions range from green to yellow, and rigid, highly sclerotized zones show up in red. A: Ventral view of the closed labellum, showing resilin-rich areas around the furca and the lip-like structure at the tip. B: Slightly opened labellum in lateral view, exposing denticles and the elevated position of the furca. C: Partially opened labellum in dorso-lateral view, exhibiting prestomal teeth and gustatory sensilla. D: Open labellum in ventral view, highlighting teeth-like structures. The full proboscis is visible at the top of each panel for orientation. In panel C, the perspective of the proboscis is rotated by 45° along its axis to show the magnified view of the labellum. Abr.: dn, denticles; dt, dorsal teeth; f, furca; gs, gustatory sensillum; pt, prestomal teeth; rsp, rasping tooth; sl, sensillum; vt, ventral teeth.

Macro and SEM imaging of tsetse proboscis interactions during feeding.

A-C: Lateral views of a tsetse proboscis piercing through a silicone feeding mat. The labellum is partially protruding through the material (B), and a prestomal tooth is visible (C). D-G: A human skin explant showing a tsetse bite site, with remnants of the proboscis retained in the tissue (F, G). H-I: Higher magnifications of the proboscis from G, showing the hypopharynx filled with metacyclic trypanosomes (H), and expelled trypanosomes adhered to adjacent regions of the proboscis (I). J: A tsetse fly feeding on cow skin; the body was removed and the sample subsequently fixed. K-L: Ultra-thin section of resin-embedded cow skin. Shown is a proboscis within the biting channel (K). Successive magnifications reveal trypanosomes, indicated by orange arrows, in the surrounding tissue (L). M-N: Overview and SEM detail of V. niloticus lizard skin with a tsetse proboscis inserted between the scales. Abr.: bc, biting channel; ce, compound eye; cs, cow tissue; ed, epidermis; fl, flagella; gs, gustatory sensillum; hr, hair root; hy, hypopharynx; lb, labium, lg, labial gutter; lr, labrum; me, metacyclic trypanosome; ml, muscle layer; mx, maxillary palp; N, nucleus; pr, proboscis; pt, prestomal tooth; ps, penetration site; sm, silicone membrane; thr, thread; tn, tendon.

Comparison of penetration and retraction forces during proboscis-substrate interaction.

Related to Figure 8. A-B: Heatmap and violin plot of penetration and retraction forces across all substrate types. C-D: Heatmap and violin plot of penetration and retraction forces across all animal samples (excluded are silicon and PDMS substrates). Statistical significance was determined using the One-sample Wilcoxon test; ****, p<0.0001.

Force measurements during proboscis-substrate interaction.

A: Example force plots of tsetse fly probing into PDMS and a hde, showing initial penetration, continued probing, and forced retraction. During probing on the hde, a drinking phase is visible. B: Visualization of the force measurement setup. A starved tsetse fly was held in a testing vial above a sample, which was mounted on a 3D-printed platform connected to a force sensor. The sample was heated to simulate host body temperature, triggering probing behaviour. C, E: Heatmaps of the penetration (C) and retraction (E) forces measured during fly probing. Each square represents a single probing event, grouped by substrate on the x-axis. D, F: Violin plots of the penetration (D) and retraction (F) forces measured during fly probing. The plots display full measurement ranges, with dashed lines marking medians and dotted lines marking quartiles. Statistical significance was determined using the Kruskal-Wallis test; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. All values for penetration and retraction forces are provided in Supplementary material 2. Abr.: cp, continued probing; csk, cow skin; dr, drinking; dsk, deer skin; fr, forced retraction; fs, force sensor; hde, human dermal equivalent; hl, holder; hse, human skin equivalent; hsx, human skin explant; ip, initial probing; lsk d, lizard skin (dorsal); lsk tr, lizard skin (tailroot); s, sample; sfm, silicone feeding mat; sl, silicon layer; ts, tsetse fly; vi, testing vial.

Quantification of liquid uptake dynamics during tsetse feeding.

All parameters were determined from flies feeding on dermal equivalents during proboscis force measurements. Shown are boxplots of the sample weight loss over time (A), the blood meal weight (B), and the blood meal duration (C) for 24 tsetse flies. Boxes span the interquartile range, whiskers the full range, and a line marks the median. In A, “total” indicates the overall sample weight loss during the experiment, derived from weight measurements by the force sensor. Evaporative loss was estimated and subtracted, yielding the rate of liquid uptake (“drinking”) by the tsetse flies. All values are provided in Supplementary material 3.