Whip spiders (Amblypygi) become water-repellent by a colloidal secretion that self-assembles into hierarchical microstructures
© The Author(s). 2016
Received: 30 September 2016
Accepted: 11 November 2016
Published: 28 November 2016
Among both plants and arthropods, super-hydrophobic surfaces have evolved that enable self-cleaning, locomotion on water surfaces, or plastron respiration. Super-hydrophobicity is achieved by a combination of non-polar substances and complex micro- and nano-structures, usually acquired by growing processes or the deposition of powder-like materials.
Here we report on a multi-phasic secretion in whip spiders (Arachnida, Amblypygi), which externally forms durable, hierarchical microstructures on the basically smooth cuticle. The solidified secretion crust makes the previously highly wettable cuticle super-hydrophobic. We describe the ultrastructure of secretory cells, and the maturation and secretion of the different products involved.
Whip spiders represent intriguing objects of study for revealing the mechanisms of the formation of complex microstructures in non-living systems. Understanding the physical and chemical processes involved may, further, be of interest for bio-inspired design of functional surface coatings.
Water-repellence is an important property for various biological surfaces, avoiding unwanted wetting, contamination, water-loss, fouling and conglutination, and permitting self-cleaning effects, locomotion on water surfaces, and plastron respiration [1–6]. While hydrophobicity by surface chemistry can achieve a maximal water contact angle (CA) of ~120°, the super-hydrophobic state (CA > 150°) is achieved by additional microstructuring of the surface . Because small water droplets form a nearly spherical shape, they roll off the surface at a tilted angle below 10° . The report of this phenomenon in plants in the late 1990s  generated enormous interest in water-repellent and self-cleaning surfaces, and led to innovations in artificial super-hydrophobic materials and surface coatings [9, 10]. However, there are many drawbacks in the production of such materials, and especially the durability of such coatings is often not satisfying .
Overview of different mechanisms that produce superhydrophobic surfaces (Water-CA > 150°) in plants and animals
lotus (Nelumbo nucifera)
wax crystals on nubby epidermis
nasturtium (Tropaeolum majus)
tubular wax crystals
damselfly wing (Calopteryx splendens)
rod-like wax crystals
sawfly larva (Rhadinoceraea micans)
wax crystals on nubby cuticle
butterfly wing (Papilio xuthus)
microstructured scale-like setae
water strider (Gerris remigis)
backswimmer (Notonecta glauca)
setae and microtrichia
springtail (Tetrodontophora bielanensis)
leaf hopper (Athysanus argentarius)
fishing spider (Dolomedes triton)
setae with lipid coating
whip spider (Amblypygi)
granulated secretion coat
Whip spiders (Arachnida: Amblypygi) are tropical or subtropical arachnids living in damp places, such as caves, leaf litter or under tree bark . Weygoldt, wjp was the first and the only one who comprehensively study the biology of these cryptic arachnids, noted a ‘clay-like’ powder on the cuticle of whip spiders . However, further data on this ‘powder’ are lacking. It has been reported that whip spiders are able to survive submerged due to plastron respiration , however the physical mechanism of plastron formation remained obscure. It was proposed that the wrinkled structure of the cuticle close to the book lungs is responsible for water-repellence and air-entrapment . However, plastron-forming structures are usually more complex and show roughness on finer length scales . Gland openings “of unknown function” have been repeatedly found on the cuticle of whip spiders [21, 22]. This may indicate the importance of secretions for waterproofing. We hypothesize that the granular coating reported by Weygoldt  is a secretion product that is responsible for the entrapment of air and a repellence of water. In the present study we sought to test this hypothesis and to reveal the fine structure and origin of this substance.
We investigated the ability to repel water and the surface structure of the carapace in the following species of whip spiders: Charinus acosta (Quintero 1983) from Artemisa-Cuba (Charinidae), Charon cf. grayi (Gervais 1842) from Negros-Philippines (Charontidae), Damon annulatipes (Wood 1869) from Durban-South Africa, and Phrynichus ceylonicus (C. L. Koch 1843) from Beliluhoya-Sri Lanka (Phrynichidae); Paraphrynus carolynae Armas 2012 from Arizona-USA, Phrynus longipes (Pocock 1894) from Peninsula Samaná-Dominican Republic, and Phrynus decoratus Teruel & Armas 2005 from Cienfuegos-Cuba (Phrynidae). This covers all extant whip spider families, except Paracharontidae, which are nearly unobtainable . Study animals were wild caught or bred from wild-caught animals, and kept in plastic- and glass terraria using standard methods . Temperature was kept constant at 26–27°C and relative humidity varied between 65 and 75%. Animals were fed every seven days with cricket nymphs (Acheta domestica) in suitable sizes.
To test the wettability of the whip spider cuticle, 30 μl droplets of tap water were dripped on the carapace (dorsal prosomal shield) from a height of 1–5 cm (depending on the size of the animal) using a micro-pipette. Tap water had the following characteristics: conductance 448 μS; GH 14°; KH 10,5; pH 8,0 NH4 < 0,05 mg/l; NO2 0,025-0,05 mg/l; NO3 1,0 mg/l; Fe 0,02-0,05 mg/l; PO4 < 0,02 mg/l; SiO2 2,0-3,0 mg/l; Mg 10,0; K 2,0 mg/l. In a second test series, two days later, droplets were carefully placed on the carapace, and we observed whether they remained attached or rolled off when the animal moved. Both nymphs and adults were tested, with 3–15 individuals per species.
For microscopy studies, pieces of freshly collected, air dried exuviae (carapace and femur leg IV) were used. To study the secretion process we studied two animals of C. acosta, with one killed (~2 min in a deep-freezer at –20°C) ~2 h after moulting, and another one ~30 h after moulting. The specimens were cut in half, and one piece was air dried and the other one was fixed in 2% glutaraldehyde solution in 0.1 M sodium-cacodylate buffer. Afterwards specimens were rinsed in buffer and postfixed in 1% osmium tetroxide for approximately one hour before rinsing 3–4 times in double-distilled water. Samples were afterwards dehydrated via acidified dimethoxy-propane (DMP) for 30 min followed by three washes in acetone. Furthermore, a freshly ablated, air dried leg of a P. longipes 6 h after moulting, and pieces of carapace and walking legs of the same individual deep frozen 24 h after moulting, were studied. For light microscopy images a multifocal stereo microscope (Leica M205 A, Leica Microsystems GmbH, Wetzlar, Germany) equipped with a camera (Leica DFC420) was used. For scanning electron microscopy, samples were sputter coated with 10 nm Au-Pd and viewed in a Hitachi S-4800 SEM (Hitachi Ltd., Tokyo, Japan) at an acceleration voltage of 3.0 kV. Samples of moulted C. acosta were sputter coated with 20–40 nm gold and examined in a JEOL IT300 SEM (JEOL, Akishima, Japan) at 20 kV.
Histological and cytological processing
Freshly moulted and inter-moult specimens of C. acosta were treated as described above, including the complete dehydration step in acetone before being embedded into Agar Low Viscosity Resin (LVR, Agar Scientific, Stanstead, Essex, UK). Cured resin blocks were sectioned at 1 μm (semithin) or 60 nm (ultrathin) on a Leica UC6 ultramicrotome (Leica Microsystems, Wetzlar, Germany). Semithin sections were stained with toluidine blue for few seconds on a heating plate at 60 °C, whereas ultrathin sections were contrasted with uranylacetate for 30 min and leadcitrate for 5–10 min. Light microscopy sections were viewed with an Olympus BX53 equipped with a DP73 microscope camera (Olympus, Tokyo, Japan). Ultrathin sections were analyzed with a Zeiss Libra120 TEM (Zeiss, Oberkochen, Germany).
Structure of the cuticle coating
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.
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
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)
Plants and animals usually acquire super-hydrophobic surface structures through growth, self-assembly of waxes, or the deposition of nano-particles (Table 1). The mechanism, we found in whip spiders, differs in that the surface structure (and resulting super-hydrophobicity) arises from an initially homogeneous secretion coat after its extrusion onto the cuticle surface. Such an additional layer of a solidified secretion crust forming complex microstructures has previously been described as so-called cerotegument in mites  and millipedes , but never from arthropods as large as whip spiders. Furthermore, the secretion processes and formation of structures have never been reported to date. We found a remarkable complexity of the resulting surface: there is a micro-roughness caused by the granules arising from assembling droplets, and a nano-roughness caused by the arrangement of nano-particles at the interface between two immiscible phases, or by droplets of a volatile component leaving dimples behind. This topography leads to the enclosure of air bubbles when wetted, which further decreases the attachment of a water droplet (Cassie state) .
Based on our microscopic observations, we propose that there are two main secretion fractions extruded separately, with one water-based phase emerging from the major gland openings, and a another phase based on an electron-denser solvent and emerging from the pore-like minor gland openings. This is indicated both by a difference in staining behaviour and the distinct cellular structures present in both types of secretory cells. However, the exact mechanisms forming the microstructures during the long curing process remain unclear, especially since the chemical identity and physical properties of secretion fractions are not known. Water may act as a volatile medium partitioning globular fractions from the insoluble phase by specific surfactants. It is also probable that exoenzyme processes play a role, which would explain the long duration of the crust maturation.
The nano-particles that assemble on the surface of the granules and the continuous film in between form regular patterns. Such self-assembly of nanoparticles at interfaces can be driven by attractive and repulsive interactions or capillary forces during evaporation of the volatile medium, as it is applied in colloidal lithography . Hierarchical globular structures with hydrophobic properties, comparable to the shape and size of granules of some whip spider species we studied (C. cf. grayi and P. ceylonicus) have previously been synthetically produced by multi-step colloid lithography . Nonetheless, the structural diversity we observed in different species of whip spiders cannot be generated by such methods. A chemical analysis and in vivo observation of secretion assembly is necessary to shed light on the underlying mechanisms involved in the formation of such complex and highly un-wettable structures as found in whip spiders. Mimicking such coatings for technical applications may not only be of interest as self-cleaning and water-repellent surfaces, but also for a broad range of hierarchically structured, functionalized surfaces with high wear resistance.
The biological function of the super-hydrophobic coating in whip spiders is not known and rather speculation. It may be related to plastron respiration during over-flooding of the microhabitat. Many whip spider species live on the ground or in caves and have been shown to stay in a close range of their resting site . Laboratory studies on the species Phrynus marginemaculatus have demonstrated that it can survive for more than 24 h when submerged in water, indicating the presence of a plastron . Due to the Cassie state, a thin air film is formed in water and may act as a physical gill. Interestingly, the tips of tubercles often stay free of secretion and are highly wettable. Such structure may stabilize an air film, similar to the recently described Salvinia-effect in swimming plants . In addition, the hierarchically-structured secretion layer may provide a self-cleaning effect, prevent bacterial adhesion, as well as play a role in coloration and camouflage. Due to its smoothness, the whip spider cuticle is highly reflecting and shiny, but matted by the secretion that optically appears similar to clay dust. Areas of different secretion amounts contribute to particular coloration patterns, blending the animal with its environment.
These observations represent an intriguing new example of a functional biological surface and may shed new light on the biology of whip spiders. Elucidating the physico-chemical processes involved could render new ideas for the development of novel colloid-generated coating techniques, in order to achieve durable films of hierarchical microstructures on various surfaces.
We thank S. Huber, M. Gamache, P. Pumberger and P. Grabowitz for providing animals, specimens and/or collection assistance. We thank the Core Facility Cell Imaging and Ultrastructure Research of the Faculty of Life Sciences in Vienna for support with the TEM and SEM imaging. We thank Dr. Anton Lamboj (University of Vienna) for determining the characteristics of the tap water used for wetting tests. We thank four anonymous reviewers, of which two provided constructive criticism and comments on an earlier submission, and two on the current submission of this manuscript.
JOW received funding from Macquarie University (Macquarie Research Fellowship).
Availability of data and materials
Representative materials from all studied animals are available at the Natural History Museum Vienna (NHMW, curator Mag. Christoph Hörweg).
JOW, MS and SNG conceived and designed the study. JOW, TS and MS performed the experiments. JOW and TS wrote the paper, and MS and SNG equally contributed in revision. All authors gave final approval for publication.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
Not applicable, because we used non-regulated invertebrates, no CITES animals or animals from protected areas.
There is supplemental material linked with this article.
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