Characterisation of a skin secretion with adhesive properties in the ground frog Eupsophus vertebralis (Alsodidae)

Some skin secretions with adhesive properties allow frogs to distract predators and escape; their nature is poorly studied. Here, we report the sticky skin secretion released by the Patagonian frog Eupsophus vertebralis when stressed. This secretion contained ~ 50% proteins spanning 25–250 kDa and required a fast setting time to turn into strong adhesive, which worked well on synthetic and biological materials. Lap-shear assays with Eupsophus glue secretion showed average shear strength of 3.34 MPa, comparable to cyanoacrylate (5.47 MPa). These properties suggest its biotechnological value for practical applications in industrial and medical sectors.

The genus Eupsophus is an endemic taxon of temperate rain forests of Chile and Argentina (Blotto et al. 2013), which includes ten species, divided into the roseus and vertebralis groups (Formas and Brieva 1992). The roseus group includes E. altor, E. roseus, E. calcaratus, E. contulmoensis, E. insularis, E. septentrionalis, E. migueli and E. nahuelbutensis, while the vertebralis group consists of E. vertebralis and E. emiliopugini (Suárez-Villota et al. 2018). During herpetological surveys on this genus, we observed that some Eupsophus vertebralis specimens released a sticky cutaneous secretion when they were suddenly provoked (Fig. 1A).
Gluey skin secretions have been reported in a few species, including some representatives from all three or-ders of extant amphibians (Evans and Brodie 1994;Tyler 2010). In general, skin secretion with adhesive properties could glue-dry leaves or debris to a predator's mouth, thus distracting the attacker and allowing the prey to escape (Evans and Brodie 1994). The distasteful and/or toxic nature of some adhesive secretions could complement the chemical defence strategy of amphibians (Phillips and Shine 2007). The most reported sticky secretion is produced by species of Notaden, a genus of Australian fossorial frogs, belonging to subfamily Limnodynastinae (Graham et al. 2016;Tyler 2010). When provoked by potential predators, these frogs release a sticky non-toxic material from their dorsal skin that contains mainly proteins with few carbohydrates (Graham et al. 2005; Gra- ham et al. 2016). On the other hand, sticky secretions that fasten the breeding pair during amplexus perform a role relevant to anuran reproduction. These secretions are produced from adhesive glands, which pertain to the wide and heterogeneous category of "breeding glands" (Brizzi et al. 2003). Interestingly, Siegel et al. (2008) demonstrated that adhesive glands in the sternum and forearm regions of male Gastrophryne carolinensis are derived from mucous glands and give negative histochemical responses to proteins.
To explore some physical-chemical properties of Eupsophus sticky secretions, we captured five E. vertebralis specimens from Llancahue, Región de Los Ríos, Chile (-39.8394, -73.1301). We carried out this procedure under the supervision and approval of the Bioethics and Biosecurity Committee of the Universidad Austral de Chile (UACh, Resolutions No. 236/2015 and 61/15) and the Servicio Agrícola y Ganadero (SAG, Resolution No. 9244/2015). Amongst these specimens, one frog released a sticky cutaneous secretion, which was collected using a sterile spatula directly from the skin and stored in ethanol (96%) at -20 °C. Subsequently, the animals were released at the collection site.

Main physical properties
Sticky samples collected from E. vertebralis skin had a fast setting time, from ~ 10 s to ~ 2 min, turning into an adhesive with similar features to silicone (Fig. 1B). Transparent flexible samples in ethanol had less adhesive capability and turned into white-yellow crystals when removed from alcohol at room temperature. A major difficulty for analysing this adhesive secretion consisted of diluting such crystals in a suitable solution. However, we adopted effectively the strategy developed by Graham et al. (2005), using 5% (v/v) acetic acid, 10% (w/v) SDS, 5 M guanidinium hydrochloride (pH 5.0) and 10 mM phosphoric acid (H 3 PO 4 ). Nevertheless, samples treated with SDS took more than 24 hr to dissolve. Interestingly, when crystals were rehydrated in water, the sticky properties of the secretory product were restored. The adhesion of these rehydrated samples worked on metal, paper, plastic and glass surfaces, retaining their properties in cold conditions ( Fig. 1C-F, tested at -20 °C and room temperature). We also tested the adherence to biological samples such as skin, bone and cartilage ( Fig. 1G-I) at room temperature.

Mechanical properties
Lap-shear tests of E. vertebralis dry secretion (rehydrated in distilled water) were performed in a microcomputer-controlled electronic universal testing machine (Model WDW-10E, MUE 10 kN, Time Group). For this purpose, pairs of poplar-wood craft sticks were lap-jointed by sandwiching a piece of rehydrated secretion between a 1 cm overlap. Ten of these test pieces were allowed to dry for two weeks and then tested in the universal machine using 1 kN static load cell and a cross-head speed of 1 mm/min, according to Graham et al. (2005). We also carried out this procedure on ten pieces for each commercial glue: (Pritt Stick-Fix ID:4595328), polyvinyl acetate and cyanoacrylate. Thus, the mean shear strength of E. vertebralis secretion was 3.34 MPa (SD = 0.98), showing bond strengths greater than dried Pritt Stick-Fix (1.63 MPa, SD = 0.87) and comparable to polyvinyl acetate (2.76 MPa, SD = 1.64) and cyanoacrylate (5.47 MPa, SD = 1.95) ( Fig. 2A).

Biochemical traits
Bradford's assay on Eupsophus secretion revealed about 50% of proteins/dry weight, as estimated in samples diluted in 5% v/v acetic acid, using the commercial Bradford Protein kit (Co. Thermo Fischer, USA) and Nano-Drop ® ND-1000 Spectrophotometer. For electrophoretic separation, dehydrated secretion was eluted in acetic acid 5% (Graham et al. 2005). We carried out several assays using elutions from 1 mg/μl to 500 mg/μl: concentrations between 10 mg/μl and 100 mg/μl gave better results. Then a fraction of 10 μl of eluate solution (10 mg/μl or 100 mg/μl) was added to 90 μl of sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE) loading protein buffer and then boiled at 95 °C for 10 min. Subsequently, 5 μl aliquots were loaded per lane in an SDS-PAGE composed of 12% running gel and 4% loading gel. Electrophoresis was run at 100-130V for 3 hr. Proteins were visualised by staining with Simple Blue Safe Stain (safe Coomassie G-250). This electrophoretic separation of the E. vertebralis secretion disclosed eight distinctive protein bands (Fig. 2B). The most abundant proteins had apparent molecular masses ranging from 150 to 250 kDa. The electrophoretic bands in this range had an odd turbulent appearance (Fig. 2B), which was more obvious at a higher dilution of the adhesive material (Fig. 2B, compare Ev and Ev1 lines).

Discussion
Our analyses disclosed adhesive properties and moderate protein concentration in secretions from South American Eupsophus species, comparable to those from Australian Notaden species (Graham et al. 2005;Graham et al. 2016;Tyler 2010). The extent of secretion release from specimens of E. vertebralis was highly variable. In fact, most of the individuals did not accomplish the "bulk discharge" described for cutaneous glands in other amphibians (Quagliata et al. 2008). Amphibian skin has greater diversity of glandular structures compared with other vertebrates, such as mucus glands, serous (granular) glands, lipid glands, mixed mucous-serous glands and specialised glands (sexually dimorphic skin glands, SDSG), representing modified mucous or serous glands (Brizzi et al. 2003). These cutaneous glands produce distinctive "bioactive" molecular classes, including proteins, biogenic amines and alkaloids, which are usually secreted by serous glands (Daly et al. 1987). Thus, proteinaceous material from E. vertebralis secretion may be physiologically discharged on to the body surface by contraction of the muscle sheaths (myoepithelia) enveloping serous or serous-derived specialised glands. In anurans, myoepithelium contraction is controlled by an adrenergic mechanism (Holmes et al. 1977). Therefore, the highly variable extents of secretion release amongst Eupsophus specimens represent graded defence responses and may depend on the perceived noxious manipulation.
A moderate concentration of proteins was found in dry Eupsophus secretion (50%) comparable to Notaden sticky secretion (55-60%; Graham et al. 2013). In other bio-adhesives from different animal sources, a diverse range of proteinaceous contents has been detected. In fact, protein concentration lower than 20% (i.e. in Holothuria;DeMoor et al. 2003) or higher than 78% (i.e. in Plethodon;von Byern et al. 2017) have been reported. Although some proteins, such as Nb-1R or Er_P1 (in Notaden and Euperipatoides secretions, respectively) are the major component of some glues and appear to be the key structural component in the adhesion, it should be stressed that similarities detected between adhesive materials from different animal sources most likely represent the outcome of convergent evolution (Graham et al. 2013). In this regard, Nb-1R and Er_P1 proteins have high MW (260-500 kDa), similar to the prevailing proteins of Eupsophus secretion (250 kDa, Fig. 2B) and all share oddly "turbulent" electrophoretic bands. Performing further electrophoretic and proteomic trials (from amino-acidic composition to increasing levels of protein structure), will allow us to establish whether such similarities are associated to comparable setting and adhesion mechanisms in these species.
Secretion from E. vertebralis showed adherence to various materials and its shear strength was comparable to commercial glues, resembling secretions with biotechnological potential collected from other amphibians (Graham et al. 2005;von Byern et al. 2017). For example, the secretion from Notaden frogs has elicited great interest for practical applications in the industrial and medical fields (Tyler 2010) and has been patented as a therapeutic adhesive derived from a natural source (Tyler and Ramshaw 2002). Indeed, Notaden frog glue has increased the strength in ex vivo-restored rotator cuff (Millar et al. 2009) and meniscus constructs (Szomor et al. 2008) from sheep. From an ophthalmological perspective, Notaden glue has proved to adhere successfully to collagen-coated perfluoropolyether lenses and debrided bovine corneas, supporting epithelial re-growth in a culture system (Graham et al. 2010). Moreover, tissues re-absorbed small pellets of glue implanted subcutaneously into mice (Graham et al. 2010). Thus, future research, like those carried out with Notaden frog glue, will allow us to know whether Eupsophus secretion could have a promising future in biomedical applications. The physical-chemical traits of this secretory product, such as its property of being preservable in solid state (dried), total recovery of the adhesion after hydration and capability of adhering to cold surfaces, represent potential advantages, suitable for the development of new biomimetic or biotechnological materials for application in the biological and industrial fields.