Materials Research Express ACCEPTED MANUSCRIPT Extracellular micro and nanostructures forming the velvet worm solidified adhesive secretion To cite this article before publication: Yendry Corrales et al 2017 Mater. Res. Express in press https://doi.org/10.1088/2053-1591/aa9940 Manuscript version: Accepted Manuscript Accepted Manuscript is “the version of the article accepted for publication including all changes made as a result of the peer review process, and which may also include the addition to the article by IOP Publishing of a header, an article ID, a cover sheet and/or an ‘Accepted Manuscript’ watermark, but excluding any other editing, typesetting or other changes made by IOP Publishing and/or its licensors” This Accepted Manuscript is © 2017 IOP Publishing Ltd. During the embargo period (the 12 month period from the publication of the Version of Record of this article), the Accepted Manuscript is fully protected by copyright and cannot be reused or reposted elsewhere. 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All third party content is fully copyright protected, unless specifically stated otherwise in the figure caption in the Version of Record. View the article online for updates and enhancements. This content was downloaded from IP address 131.172.36.29 on 12/11/2017 at 09:08 Page 1 of 17 3 4 Yendry Regina Corrales-Ureña1, Angie Sanchéz1, Reinaldo Pereira1, Klaus Rischka2, Thomas. Kowalik2and José Vega-Baudrit1,3. 5 6 7 8 9 10 1. National Laboratory of Nanotechnology, Costa Rica, LANOTEC-CENAT-CONARE. 1.3 km north from the USA embassy, San José, Costa Rica. 2. Fraunhofer Institute for Manufacturing Technology and Advanced Materials IFAM, Adhesive and Polymer Chemistry. Wiener Straße 12, 28359 Bremen, Germany. 3. National University of Costa Rica, UNA,Heredia, Costa Rica. 11 Corresponding author: [email protected] us cri pt 2 Extracellular micro and nanostructures forming the velvet worm solidified adhesive secretion 1 12 Abstract 14 The onycophoran Epiperipatus hilkae secrets a sticky slime that solidifies almost immediately upon 15 contact with air and under high humidy environmental condition forming a glassy like material. The 16 general adhesive biochemical composition, the releasing and hardening mechanism have been 17 partially described. In this study, the structural characterization of the extracellular microstructures 18 and nanostructures forming the solid adhesive of the secretion from Epiperipatus hilkae velvet worm 19 is presented. The adhesive secretion is formed by macro-threads, which, in their solid state, are 20 composed of globular particles approximately 700 nm in diameter that are distributed homogeneously 21 throughout the matrix surface, and nanoparticles approximately 70 nm in diameter that self-assemble 22 forming fiber-like structures. Nanoparticles with non roundish forms are also observed. These 70 nm 23 particles could be associated to proteins that form high density coverage films with low roughness; 24 suggesting the formation of two dimensional ordered films. A crystalline and an amorphous phase 25 composes the solidified secretion. The glassy or viscoelastic properties depend on the time in contact 26 with air before being adhered to a solid surface and/or the mechanical stimulus; suggesting a key role 27 of the drying on the hardening process. 28 Keywords: adhesive, natural, onycophoran, velvet worms, nanoparticules, protein, crystals. 30 31 dM pte ce 29 an 13 Introduction Advances in nanotechnology and molecular biology have promoted material development using bioinspired approaches [1]. Nano-defined self-assemblies derived from biological systems have been Ac 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 AUTHOR SUBMITTED MANUSCRIPT - MRX-105697.R1 32 used as an inspiration for the innovative development of materials, such as bio-adhesives that could 33 work efficiently in water using cross-linked non-toxic components. Some animals and plants produce AUTHOR SUBMITTED MANUSCRIPT - MRX-105697.R1 adhesive secretions for prey capture, defence, prevention of dehydration, and camouflage, among 35 other things; and have been used as the inspiration for the design of new adhesives to be applied in 36 the medical, bio-electronical, textile and cosmetics industries [2,3]. Recent examples include mussels, 37 frogs, ivy plants, sandcastle worms, geckos, sea cucumbers and tubeworms [4-12]. Each organism 38 has its own features and the physicochemical characterization of biological adhesives is challenging. 39 In general, these secretions are composed mainly of mixtures of proteins, carbohydrates, surfactants, 40 peptides, water and some ions like Ca2+. Natural adhesives usually consist of complex biopolymer 41 blends, forming in many cases extracellular nanometric structures that play a key role in the adhesion 42 mechanism. Some of the functions of the extracellular nanostructures are attributed to the 43 enhancement of energy dissipation, as it is frequently found in climbing animals that produce 44 fibrillary structures. These structures are thought to be responsible for a mechanism analogous to the 45 molecular stretching of polymeric chains and also, through their nanostructures, to influence the 46 contact points with the target surfaces to minimize crack length and propagation [10]. 47 The onychophorans, or velvet worms, live in tropical areas such as Costa Rica. These are small 48 terrestrial invertebrates, similar in appearance to caterpillars, which capture their prey by means of a 49 sticky secretion that is launched through passive oscillation. The viscous slime is expelled by dual 50 high-speed, and force flexible, tubes or cannons called oral papillae; the muscular action produces a 51 swinging movement of the adhesive-spelling organs. In these cannons the slime is squirted and under 52 an oscillation process the material is expelled like a jet, in small drops, some inches away [13]. 53 The velvet worms in Costa Rica belong to the family of Peripatidae, which has a circumtropical and 54 subtropical distribution, including the African continent (tropical West Africa), Southeast Asia and 55 tropical America. Their liquid ejection mechanism operates on a micrometric scale and is a model for 56 the micro and nanofabrication of fibres. The secretion contains several substances of high commercial 57 and biotechnological potential: low-structure adhesive proteins, molecules that can recognize sugars 58 (lectins), surfactants, polyphenols, and peptides with potential antimicrobial properties. The general 59 composition of the slime has been characterized. It is composed mainly by 90 wt. % of water and 60 10 wt. % of proteins, carbohydrates and lipids. Small peptides are also present in the adhesive 62 63 64 us cri an dM pte ce 61 pt 34 secretion [17]. Despite having been studied, by different authors [13-21], only recently the mechanical and morphological properties of the velvet worm secretion was partially described; suggesting a mechanically responsive material that can be drawn into stiff fibers that can be dissolved and regenerated [22]. Ac 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 2 of 17 65 For understanding the hardening mechanism, in this contribution we describe the morphologies of 66 the extracellular structures in the solidified secretion by atomic force microscopy (AFM), optical and Page 3 of 17 polarized microscopy, transmission electron microscopy (TEM), contact angle and the chemical 68 composition using through Fourier transform infrared spectroscopy (FT-IR), energy dissipation X- 69 ray diffraction (EDX) characterization techniques. 71 us cri 70 pt 67 Materials and methods 72 Sample collection: specimens were collected in San Ramón Costa Rica in their natural habitat. 74 Autoclave silica sample holders were used for collecting the samples in situ. Five samples of two 75 specimens were analyzed. The procedure for collecting the adhesive used was previously described 76 by Concha et al. [13]. The pristine sample without any treatment was analyzed by an optical 77 microscope Motic BA 410, a polarized microscope BA 300Pol and a confocal microscope Leica 78 DCM 3D. The environmental conditions during the measurements not made in vacuum were: 79 temperatures of 22°C and an average of 85 % relative humidity. The samples were storage under 80 aseptic conditions and imaged by AFM the same day for avoiding degradation. However, the material 81 maintained their aspect and size during several days under storage at ambient conditions. To 82 determinate the influence of the drying, the samples were put in an oven at 95°C for 30 min. The 83 surface topography was analyzed using an AFM Asylum Research operated in the tapping mode in 84 air. Silicon probes (model Tap150Al-G, back side of the cantilever covered Al) with resonance 85 frequencies of 150 kHz and force constant of 5 N/m were used. Slices of the solidified adhesive were 86 cut using a new and clean glass knife forming a powder and mounted onto a 400 mesh 87 copper/palladium carbon coated grid.The TEM images were obtained on a JEOL JEM2010 88 Transmission Electron Microscope/EDX set at 120 kV. Several samples were prepared and analyzed: 89 A1: sections of the solidified secretion, A2) A1 after adding a drop of 5 μl miliQ water on top of 90 surface and let it dry. Next, they were fixed with a solution of 2.5 wt.% glutaraldehyde in 1mM 91 phosphate buffer to preserve the ultrastructure, B1 and B2 were prepared as sample A1 and A2 92 without fixative. The purpose of A and B preparations was to have a clean EDX analysis to determine 93 the possible composition of the structures forming the secretion and avoiding changes due to drying 95 96 97 dM pte ce 94 an 73 under the TEM vacuum chamber. FT-IR spectra were measured on a Bruker Vertex 70 with an IR Scope II extension. The measurements were performed in ATR technique (Attenuated Total Absorption in the IR Scope II extension, standard Bruker ATR-lens with Germanium crystal, one reflection, 32 µm tip diameter). ATR technique allows surface sensitive measurements with Ac 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 AUTHOR SUBMITTED MANUSCRIPT - MRX-105697.R1 98 information depths up to 1.5 µm. The information depth depends on the surface roughness, refraction 99 index of the used crystal and samples; among other variables and the result is an average value. Single AUTHOR SUBMITTED MANUSCRIPT - MRX-105697.R1 measurement parameters were a scan number of 64, a resolution of 2 cm-1 and a background 101 measurement against air. Mapping mode was performed with the same parameters as automated 102 measurement. Data search was performed with the Bruker own software OPUS on base of own, 103 Merck and Sadtler data bases. The apparent contact angles were measured using a goniometer 104 (OCA15 Plus, Dataphysics Instruments, Germany) by sessile drop technique and MiliQ grade water 105 was used as probe liquid; the volume of the drops was constant (10 μl) for each measurement at a 106 temperature of 22˚C. The contact angle values reported are an average value of at least three separate 107 drops on different substrates area. The recorded images were analyzed by SCAN 20 Dataphysics 108 software. 109 Results and discussions 110 In order to understand the adhesion principle of the onychophoran slime, a morphological 111 characterization was performed. Secreted material was deposited by the specimen directly onto a 112 clean silica substrate; possible external residues of soil could be correlated to the residues in the 113 papillae. The samples were collected in San Ramón Alajuela, Costa Rica, in their natural habitat 114 inside the forest. The specimen was a Epiperipatus hilkae [14], figure 1 A. Figure 1 B shows a light 115 microscopy image of the biofilm secreted. The solidified adhesive formed macro-meter linear threads. 116 Figure S1 shows fiber like formation inside the biofilm and in other cases some particles like 117 structures. The adhesive could be described as translucent material which spontaneously adhered to 118 glass, figure 1 B, and it is a glassy-like solid. The secretion solidified in approximately 10 seconds 119 after expelling and being in contact with the substrate surface. A whitish viscoelastic material was 120 obtained when the material was accumulated at the bottom of the substrate; not having time to expand 121 through the surface, as it can be seen in figure 1 C. In that case, it remained in a viscoelastic state 122 even for 15 days until it was mechanically mixed and stretched with the metallic tweezers several 123 times; producing the hardening of the material. ce pte dM an us cri pt 100 Ac 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 4 of 17 124 125 Figure 1. A) Photograph of the onychophoran specimen. B) Light microscopy image of the secretion 126 on a silica substrate, 10 X C) Photograph of the whitish viscoelastic material. Page 5 of 17 For determining the role of the drying in the hardening mechanism, a piece of the material was cutted 128 with a metallic blazer and placed on top of a glass substrate. A drop of water was added on top of 129 the solid material and this was immediately contracted in a roundish form. A plastic pipette was used 130 to apply force and deform it, but this could not be easily elongated. Next, the water was removed by 131 capillarity using a paper towel and the material was in a semi-dried state. In that moment the material 132 could be deformed preferentially in a fiber like structure; more in the Y edge than in the X edge, as 133 it can be seen in figure 2 A and B. 134 viscoelastic a glassy like material. This cycle could be repeated several times (supplementary video 135 V1). It suggests that the material could be contracted inside the reservoir when is in a viscous aqueous 136 suspension (90 wt.% water). After a mechanical stimulus by the papillae oscillation and being 137 expelled it can be elongated when it is in a semi-dried state; molding the form of the prey before 138 changing to a high strength glassy material. Bauer et al reported stiffness of the solidified secretion 139 in the range of silkworm silk and nylon [22]. This results suggested the key role of the drying in the 140 assembly of the material. However, further studies are necessary to determine the role of a possible 141 chemical reaction, complex formation, etc. us cri pt 127 143 144 145 146 pte dM an After few seconds or mixturing, it changed their state from Figure 2. Solidified secretion A) under water B) stretched after drying. ce 142 The threads were analyzed by a confocal microscope to determine their thickness. Figure S2 and S3 shows a broad difference between the borders and center of the thread; showing features of lamellar structural layers. The thickness of the threads was in the µm range. An example of the cross-section Ac 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 AUTHOR SUBMITTED MANUSCRIPT - MRX-105697.R1 147 is shown in figure S2 and S3 C; an average of 6.5 µm height was determined. The glassy coating 148 formed with the secretion changed the silica surface to more hydrophobic domains; the value of the 149 contact angle between the surface and a drop of water was 80±5°; figure S4. us cri 150 Figure 3. Optical microscope images A) 10 X B) 40 X of the solidified secretion on glass substrates. 152 Figure 3 A shows a fractal like structure inside the matrix. Figure 3 B shows fibers like structures (e.g 153 red arrows) decorated by drops (blue arrow). Figure 4 A to D shows the polarized microscopy images 154 of the material. Figure 4 shows the presence of birefringent phase; showing molecular order [23]. 155 Two kinds of structures were observed by the polarized light microscope: fibers, figure 4 A and B, 156 and some crystal like structures, figure 4 C and D. However, not all the threads analyzed presented 157 a birefringent phase, as it can be seen in figure S 5 A and C. Further studies are necessary to determine 158 if the birefringent material is due to crystallization of the proteins, salts or material contained 159 previously in the reservoir as liquid crystals [22]. The proteins could self-assemble into a variety of 160 states: crystals, dense liquid phases, gels, fibers, and amorphous aggregates, two different states: 161 crystals and aggregates and even two different crystals forms [25]. ce pte dM an 151 Ac 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 6 of 17 pt AUTHOR SUBMITTED MANUSCRIPT - MRX-105697.R1 us cri an dM 162 Figure 4. Polarized optical microscope images of the solidified secretion A) 10 X B)40X C) 10 X and D)40 X. 165 The solidified secretion was characterized by FT-IR spectroscopy without any disturbance of the 166 original secretion after settling, as it is shown in figure S6. Figure S6 shows some examples of the 167 areas under analysis highlighted with different colors. The spectrum obtained for each area was 168 plotted with the same color as the one assigned for the area. All the areas presented similar absorbance 169 peaks. The spectrum shows a broad band between 3000 and 3800 cm-1 from the OH symmetric and 170 asymmetric stretching modes and a N–H stretching of the protein backbone. The peak at 2918 cm-1 171 corresponds to CH stretching. The peak at 1630 cm-1 is assigned to the amide I region and the 172 absorption at this wavelength is correlated with a β-sheet secondary structure. The peak at 1447 cm-1 173 corresponds to CH2/CH3 deformation. The peak at 1540 cm-1 corresponds to amide II and that at 1240 174 cm-1 to amide III [25]. The peak at 1050 cm-1 is associated with polysaccharides [17]. The results 176 177 ce 175 pte 163 164 agreed with the composition reported previously by Benkendorff et al. for the Euperipatoides kanangrensis. They described the secretion as a proteinaceous-based slime that also contains carbohydrates [17]. Amyloids β-sheet fibrillary proteins show a maxima absorption between 1611 Ac 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 AUTHOR SUBMITTED MANUSCRIPT - MRX-105697.R1 pt Page 7 of 17 178 and 1630 cm-1 [25-26]. The possible fibrillary protein structure is consistent with the microscopy 179 studies, which also identified fibrillary networks. Unstructured proteins forming the slime of the 180 Australian onychophoran Euperipatoides rowelli and β-sheet structures of the onychophoran AUTHOR SUBMITTED MANUSCRIPT - MRX-105697.R1 Macroperipatus geagy from Colombia have been reported [27-28].The proteins may have changed 182 their conformation due to drying, and in further studies the FT-IR results will be compared with the 183 non-solidified adhesives [29-30]. Furthermore, Baer et al. showed clear differences in protein 184 molecular weight and patterns between families, which could influence in the secondary structures 185 formed [8]. 186 TEM images of the solid material are shown in figures 5, sample prepared as described for A1. These 187 material contains a fiber-like structures with diameters of 51±10 nm, as it can be seen in figures 5 A. 188 Figure 5 B shows a material with a non define structure and apparent high roughness. Figure 5 C 189 shows a micrometer fiber formed by smaller fibers with an average diameter of 68±8 nm. Figures 5 190 D and E show more electro-dense globular nanostructures with diameters of 118 ±28 nm. The 191 difference in electro density suggests a different chemical composition in comparison to the 192 surrounding matrix. Figure 5 F shows material forming patterns and an amorphous material. This 193 molecular order could be correlated with the polarized microscope images showing a birefringent 194 phase. 196 197 ce 195 pte dM an us cri pt 181 Figure 5. TEM images of the solid slime secretion A)8000 B) 8000 X C) 800 X D) 6000X E) 40000X F)40000X. Ac 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 8 of 17 198 Figure 6 shows a more detailed images of the nanofibers. These fibers are organized in regions with 199 different assembly directions; showing a conformation that could be correlated to a grain boundary; Page 9 of 17 giving more resistance to applied forces in different angles. A more electrodense material with 201 roundish and non-roundish morphology is imaged on top of the fibers, Figure 6 A, B, D and S6. dM an us cri pt 200 203 pte 202 Figure 6. TEM images of the solid slime secretion A)1500 X B) 2500 X C) 4000X D) 5000 X 205 The substrate material was submerged in water for 4 hours to determine the stability and to elucidate 206 the non soluble structures inside the matrix. Figure S7 A shows the thread which was partially break 207 208 209 210 ce 204 it in pieces and/or dissolved; the fractal like structure and globules with similar sizes were observed over the silica surface, figure S 7 B. Figure 7 was prepared as described for A 2. Figure 7 A shows fractal structures bounded to a fibre like structure. Figure 7 B shows a crystal like particles, that could be correlated to the material imaged by polarize light microscopy. Figure 7 C shows globular particles Ac 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 AUTHOR SUBMITTED MANUSCRIPT - MRX-105697.R1 211 in the order of 100 nm range. Figures 7 D-F show globules with 450 ± 180 nm size bounded between 212 them by a fiber like structure; similar in size to the imaged by optical microscopy and imaged by AUTHOR SUBMITTED MANUSCRIPT - MRX-105697.R1 AFM. Figure 7 F shows a globules interconnected by fibers. On top of the globules smaller particles 214 are imaged. The results suggest that the globules, crystals, fibres and fractal structures are not an 215 artefact from the vacuum drying. The adhesive is partially composed of lipids and/or proteins that 216 can be aggregated and form these macromolecular structures [17,22,32]. dM an us cri pt 213 217 Figure 7. TEM images of the solid slime secretion A)300 B) 8000 X C) 6000 X D)1200X E) 2500X 219 10000X 220 Similar images were obtained after preparing the samples by A and B procedures. However, for EDX, 221 preparation B was preferring not to have any contamination from additional reagents. EDX analyses 222 of different areas, that were considered representative of the solid adhesive, were conducted to give 223 a rough overview of the elemental composition of the solidified adhesive [34-36]. The elements 224 carbon (C), oxygen (O) and nitrogen (N) had the highest counts in the spectrum, followed by sulphur 226 227 228 ce 225 pte 218 (S) and silicium (Si),magnesium (Mg). Calcium (Ca), phosphorus (P), (Al) and potassium (K). The Cu, Si and Al could be related to the TEM grid in concentrations lower than 0.1 at %, according to the values obtained when the grid without the sample was analyzed. The Si concentrations of 2 at. % and Al at 0.2 at. % could be related to the impurities from the substrate or soil, but all the samples Ac 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 10 of 17 229 and areas analyzed presented Si; suggesting this element as part of the chemical composition of the 230 slime. The globules presented mainly high concentration of C, N, O and S and Si. Graham et al. Page 11 of 17 suggested that disulphide bonding is a feature common to salamander, frog and onychophoran 232 secretions [7]. The crystal like structures, figure 7 B, presented higher concentration of C, N, O, Cl 233 and K. Table 1 summarizes the results. Figure S 8 shows an example of the spectra obtained by EDX. 234 Further biochemical characterization is necessary to determine the composition of the globular 235 structures, the crystals, the nanoparticles and the fibers; and to correlated them with the role in 236 adhesion and mechanical properties, but the scope of this research is focus on the general morphology 237 presented in the dried state. 238 Table 1. TEM/EDX analysis of the velvet worm slime. Concentrations given in at%. Traces of [N] [O] [Al] [Si] [S] General 48 6,6 41.8 0.2 2.0 ND Globules 69.1 21.4 9.1 0.1˂ 0.1˂ 0.1˂ Crystal like 66.3 24.9 8.3 ND ND 0.1˂ [K] [Ca] [Cu] [Mg] [P] 0.1˂ 0.1˂ 0.1˂ 0.1˂ 0.1˂ 0.1˂ 0.1˂ 0.1˂ 0.1˂ 0.1˂ ND ND 0.3 0.2 ND ND ND ND elements ˂0.1 were labelled as ˂0.1 at% due to detection limit of the technique. 240 dM 239 [Cl] an [C] Sample us cri pt 231 *ND: not detected 242 AFM imaging under laboratory conditions revealed nanoparticles with a spherical morphology that 243 were separated into two groups according to their diameter. Those in the first group, called “globules”, 244 have a circular shape with a diameter of 660±109 nm, as it can be seen in figure 8 A and B. Those in 245 the second group have a diameter of 83 ±18 nm and are homogeneously distributed inside the matrix 246 and assemble in a fibre-like structure, figure 8 D-F. Figure 8 C shows μm length fibers that could be 247 correlated with the fibers showed in figure 7 A. The nanometric particles arrangement observed in 248 figure 8 D could suggest an organization in a 2D crystal like structure (XY); forming a high density 249 coverage film. Some proteins as hydrophobin can form crystalline structure and organize 250 preferentially in 2D arrangements [37]. Figure S 9 shows an AFM cross-section of the globular 251 particles that are more exposed to the surface; the nano-roughness produced by the nanoparticles and 253 ce 252 pte 241 the amorphous matrix is 2.9±0.8 nm. Ac 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 AUTHOR SUBMITTED MANUSCRIPT - MRX-105697.R1 AUTHOR SUBMITTED MANUSCRIPT - MRX-105697.R1 an us cri pt 254 255 dM 256 Figure 8. AFM height images of the dried adhesive at A) 20 µm X 20 µm B) 10 µm X 10 µm C) 258 4.1 µm X 4.1 µm D) 2.6 µm X 2.6 µm. 259 The AFM and TEM results suggest an adhesive that is formed by roundish globules and smaller 260 particles that self-assemble in fibre-like structures. The sizes cannot be directly compared due to the 261 different conditions used by the TEM and AFM analyses. Figure 9 and S D shows the top surface of 262 the called “globules “. In one of the cases the surface is smooth, figure 9 A, and heights of 25 ±8 nm 263 and a second globule with smaller particles on top with sizes of 62 ± 18 nm between the base and 264 the upper part. A fiber like structure is connect to this globule, showing similar to the images showed 265 in figure 7 F. Figure S 10 A shows similar nanoparticles forming particles in the material before 266 adding water. The phase image shows a contrast where the particles are located, figure S 10 B. 267 Therefore, it shows the nanoparticles forming globules and fibres. Phase changes could be associated 269 270 ce 268 pte 257 with differences in composition, adhesion, friction, and viscoelasticity properties, with respect to the surrounding matrix [38]. Figure S 10 C and D shows agglomeration of this globules after adding water. Globular organic nanoparticles have been observed in the adhesives of several marine animals, Ac 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 12 of 17 271 including polychaetes, mussels, barnacles and sea stars as well as in terrestrial plants such as ivy. The 272 reported sizes are 50–100 nm and it is suggested that these form building blocks, similar to the AFM 273 results obtained in this study. The functions of the nanoparticles in other natural adhesives have been Page 13 of 17 attributed to increased strength, stiffness and toughness by preventing cracking formation and 275 propagation and increasing the surface contact between the adhesive material and the substratum [12]. 276 Bauer et al. suggested that this nanoparticle could form fibers and agglomerate in contact with water 277 [22]. This self-assemble mechanism could be the responsible for the stretching and contracting of the 278 material in a macroscale due to the changes at nanoscale. The material mechanically mixture with 279 the tweezers before hardening showed fibers and more random agglomerates of the particles, figure 280 S11. us cri pt 274 282 pte dM an 281 Figure 9. AFM height images of the dried adhesive after contact with water A) 2,5 µm X 2,5 µm C) 284 3,2 µm X 3,2 µm. B and D phase images of A and C respectively. 285 286 287 ce 283 In other natural animal adhesives, lipids have been associated with the prevention of slime adhesion to the secretory glandules and excretory pathway [13] and also with the function of displacing water from the surfaces to improve the adhesion of proteins [33]. Bauer et al. suggested the role of the Ac 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 AUTHOR SUBMITTED MANUSCRIPT - MRX-105697.R1 288 lipids to prevent premature protein self- assembly before the mechanical stimulus [18]. AUTHOR SUBMITTED MANUSCRIPT - MRX-105697.R1 pt 289 290 To determinate the possible changes of the surface due to drying without mechanical stimulus, the 292 sample was heated at 95°C for 1 hour. Figure S 12 A and B show the thread before and after drying. 293 The thread remained with similar sizes and no crystalline phases were formed, figure S 4 C. The 294 nanoparticles and globules were observed by AFM in the surface and some changes related to a more 295 elongated morphology and agglomeration, figure S 12 B. However, it suggested the stability of the 296 material. 297 Conclusions 298 In summary, we described the morphology of the solidified velvet worm secretion at environmental 299 conditions of relative humidity 85 % humidity, 22 °C and after contact with water. The solidified 300 secretion contracts under water and it can be stretched in semidried state. Extracellular nanostructures 301 such as nanoparticles of 70 nm nanoparticles that can organize in fiber like structures and 2D low 302 roughness films, particles with approximately 0.7 μm diameter, micro meter length fibres and 303 amorphous matrix forming the solidified adhesive secretion of the onychophoran Epiperipatus hilkae 304 were observed on the surface. The material presented a birefringent phase with different crystal and 305 fiber like structures. After being in contact with water the materials suffer a change; showing higher 306 density of globular particles connected by a fiber like material. The chemical composition is based 307 on proteins and carbohydrates and contains traces of metals such as Na, K, Mg, Ca, Al, and Si. 308 In addition, physicochemical studies are necessary to understand o the correlation between the 310 311 an dM pte ce 309 us cri 291 structures and the mechanical and rheological properties to synthesize a high strength new bioinspired adhesive that could be hardened in humid environments and applied in biomedical fields. This mechanical material could be used in the future not only as a universal adhesive because it adheres to Ac 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 14 of 17 312 plastic, metal and glass and biological material but also as material for biological actuators due to 313 mechanical response to the dry and humid state. Page 15 of 17 Acknowledgments 315 The authors would like to thank Dr Orlando Argüello-Miranda for his contributions to the 316 development of the topic of “nanobiodiversity” in Costa Rica and to Prof. Dr. Pedro León for sharing 317 his knowledge related to the velvet worms. 318 References 319 320 321 322 1. Grunwald I, Rischka K, Kast SM, Scheibel T, Bargel H (2009) Mimicking biopolymers on a molecular scale: nano(bio)technology based on engineered proteins. Philosophical transactions Series A, Mathematical, physical, and engineering sciences 367 (1894):1727-1747. doi:10.1098/rsta.2009.0012 323 324 2. Shao H, Bachus KN, Stewart RJ (2009) A water-borne adhesive modeled after the sandcastle glue of P. californica. Macromolecular bioscience 9 (5):464-471. doi:10.1002/mabi.200800252 325 326 327 3. Winslow BD, Shao H, Stewart RJ, Tresco PA (2010) Biocompatibility of adhesive complex coacervates modeled after the sandcastle glue of Phragmatopoma californica for craniofacial reconstruction. 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