Structure of the cuticle coating
We found that the whip spider integument is covered with a crust of a solidified secretion that forms globular microstructures (granules) with a diameter of 0.3–5.0 μm. Granules may exhibit a coat of assembled crystal-like nano-particles (Figs. 1, 2 and 3). The granules have a rough surface structure that is highly species-specific (Fig. 3). The secretion is present on all body parts, except regions situated close to and at joint membranes and segment borders, the distitarsi and pretarsi of legs, parts of the chelicerae and the eyes. It may also be absent on the tips of tubercles that widely cover the dorsal cuticle of the carapace and parts of the legs (except in C. acosta).
TEM images of granules in C. acosta shows a multi-layered structure (Fig. 4m). The bulk of a granule is formed by a homogeneous material. It is covered by a porous crystal-like layer of assembled nano-particles. The outermost layer is thin and irregular and composed of a loose granular substance. All these substances are rather electron-dense. SEM observations of fractured cuticles in C. cf. grayi and D. annulatipes (Fig. 6) show that the inner material appears always homogeneous, and that species-specific differences in granule morphology are the result of the specific structure of the second crystal-like layer. The uppermost layer was not visible in SEM images, and may be a fluid.
Secretion
A high amount of secretory cells is present in the epidermis, with two main types of vesicles (Figs. 4a and 5a–b): (1) spherical vesicles whose content is highly stained, and (2) large non-stained vesicles, presumably containing water (or an aqueous solution), and therefore called ‘vacuoles’. Both vesicle types are highly reduced in number and size after the formation of the secretion coat, as is the overall thickness of the epidermis (Fig. 4c). The deposited secretion crust exhibits similar staining properties as the content of the spherical vesicles, indicating that these bear the material that forms the later coat. However, the spherical vesicles do not retain their shape and are not secreted as ready-made granules. Instead, they are fusing with the vacuoles giving off a non-soluble granular substance (Fig. 5a–d).
Two types of gland openings are evenly distributed on all parts of the cuticle that eventually bear the secretion layer (Figs. 2 and 5f). The first (major gland opening) is a slit of about 4 μm length flanked by two cuticular lips (Fig. 5g). Underneath there is a cell with a highly specific microstructure (Fig. 5e, h), which includes a large lumen that is penetrated by a central tubular structure and surrounded by microvilli. The microvilli emerge from the lateral cell membranes into the lumen and their density increases gradually towards the gland opening (Fig. 5h–i). Thus there is a concentration gradient of the solution stored in the vacuole, with increasing staining affinity towards the gland opening (Fig. 5h–i). The stainable compounds come from small spherical vesicles assembling in the distal part of the cell (Fig. 5e, h).
The second (minor gland opening) is a simple pore with a diameter of about 1 μm. The pore is connected to a cylindrical lumen that contains a slightly stained isotropic substance in which highly stained nanoparticles are dispersed (Fig. 5j–k). The surrounding cells exhibit many membrane folds within the cytoplasm, indicating high synthesis activity.
Additionally, there are pore canals (diameter of ~50 nm) (Fig. 5l), scattered throughout the cuticle. In fractures of exuviae of D. annulatipes these contain an isotropic material (Fig. 6b). Such pore canals are typically present in arthropod cuticle and involved in the secretion of epi-cuticular hydrocarbons [15, 23].
Post-secretion dynamics and self-assembly
The cuticles of freshly moulted C. acosta and P. longipes show no granular structures, and their bare cuticle is rather smooth on a microscopic scale (Figs. 2 and 4d). Emerging secretion was found in some places (Fig. 4e), with a rather homogenous structure, except for some holes and dimples resulting from bubble like enclosures (Fig. 4h). In an individual of C. acosta killed 30 h after moulting, the secretion coat was evenly spread on the carapace and legs and exhibited globular structures apparently separating from the continuous film, and dimples left behind by the evaporation of a volatile compound (Fig. 4f, i–j). The final crust, as observed in an exuvia, showed a highly elaborate structure with rather similarly sized and evenly distributed granules covered by a crystal-like layer of assembled nanoparticles (Fig. 4g, l).
Samples from an individual of P. longipes, frozen 1 day after moulting followed by air drying, exhibited a thin layer of secretion on the cuticle, containing small crystals (diameter ~50 nm) and solidified droplets (diameter ~50–100 nm) (Additional file 1: Figure S3,B–D). Droplets arise from the colloidal phases of a solidifying secretion, emerging from the major gland opening, and a partly volatile secretion, emerging from the minor gland openings, that leaves dimples after evaporation (Additional file 1: Figure S3, B–D).
Once hardened, the secretion forms a stable crust that cannot be dissolved in water or ethanol. The crust can be removed in part by scratching; however, total abrasion is prevented by the tubercular structure of the cuticle.
Wettability of the whip spider cuticle
In all species tested (completely hardened surface), water droplets dripped onto the whip spider carapace roll off (Fig. 2, Additional file 2: Video S1). Water droplets directly ejected on the carapace formed spherical shapes (Figs. 1 and 3) and could only be held in place, when placed in the dimple of the mid carapace. Movement of the animal induced the droplet to roll off, except in some individuals of C. cf. grayi and P. longipes. Wettability of the smooth tips of tubercles is higher; hence the droplet may contact only these parts, indicated by a visible (reflecting) air film in between and also by a partly deformation of the droplet near the contact to the solid surface. In contrast, an individual of P. longipes was highly wettable (total spreading of the water droplet) some hours after moulting, showing that super-hydrophobicity is caused by the secretion crust formed 1–2 days after moulting (Fig. 2, Additional file 3: Video S2).
Additional file 2: Video S1: Phrynus decoratus showing super-hydrophobicity (playback speed: 50% of real time). (WMV 1549 kb)
Additional file 3: Video S2: Wettability of the cuticle of Phrynus longipes some hours after moulting and between moultings (playback speed: 50% of real time). (WMV 2236 kb)