**2.3 Microscopy**

246 Biomaterials – Physics and Chemistry

immobilise most organisms with which they come into contact (VandenSpiegel & Jangoux, 1987). The tubules, once expelled, are immediately adhesive on contact with a solid surface (VandenSpiegel & Jangoux, 1987), such as the exoskeleton or skin of a predator. Crabs, molluscs and sea stars can stimulate tubule expulsion, and the tubules stick to these species. This adhesion happens entirely under water, and does not need the mixed environment of

Sticky tubules are found only within the family Holothuridae within the order Aspidochirotida, and mostly in the genus Bohadschia and the genus Holothuria. Various authors have described the ultrastructure of the tubules, especially for *H. forskåli* (Lawrence, 2001; VandenSpiegel & Jangoux, 1987; VandenSpiegel et al., 2000), as well as their expulsion and release (Flammang et al., 2002) and the timeframe of regeneration (Flammang et al.,

Flammang and Jangoux (2004) suggested, from the differences in the surface (adhesive) protein types and compositions in *H. forskåli* and *H. maculosa*, that the adhesion proteins and mechanism may differ between species. Other studies showed that adhesive strengths varied between species, with the adhesion in *H. leucospilota* being several times greater than for six other species (Flammang et al., 2002). A limited number of studies have probed the mechanism of adhesion, focusing on *H. forskåli* and *H. leucospilota* (De Moor et al., 2003; Müller et al., 1972; Zahn et al., 1973). These studies have shown that best adhesion is found at temperatures, salinity and pH similar to those found in the marine environment in which the organism flourishes, and is most effective with hydrophilic surfaces (Flammang et al., 2002; Müller et al., 1972; Zahn et al., 1973). Increasing concentrations of urea led to a loss of adhesion, suggesting that native protein structure(s) or interctions(s) may be required for effective bonding (Müller et al., 1972). Later biochemical studies have also suggested that

In the present study, we have extended the information on Cuvierian tubule adhesion. In this study we examined the tubules of a different species, *H. dofleinii* Augustin, 1908. We have examined the distribution of the adhesive substance on the surface of expelled tubules, along with the molecular weights and amino acid compositions of its main protein components. We have estimated the strength of adhesion of *H. dofleinii* tubules to different substrata, and examined the effects of salinity, pH, ionic strength and denaturants on the

Individual *H. dofleinii* were obtained from shallow subtidal seagrass banks in Moreton Bay, Queensland, at a depth of about 1-2 metres at low tide, close to the western side of Stradbroke Island (153 26.4' E 27 25.13' S to 27 25.68' S), and were held for up to 5 days prior to use in filtered, recirculating seawater tanks at 21.5 – 22 C. The identification of the animals was based on morphology, spicule shape and size and 18S-RNA sequencing (Peng

To collect expelled Cuvierian tubules, *H. dofleinii* individuals were held and gently stimulated underwater until tubules were expelled. Immediately after expulsion, a tubule was individually collected using polytetrafluoroethylene-tipped forceps, and was allowed

the intertidal zone where many of the other potential adhesives are sourced.

the adhesive mechanism involves protein components (DeMoor et al., 2003).

2002; VandenSpiegel et al., 2000).

adhesive properties.

**2. Materials and methods 2.1 Collection of materials** 

& Skewes, unpublished data).

**2.2 Sample preparation** 

To look for the presence of glycoprotein on the surface of expelled tubules, samples were treated with fluorescently-labelled lectins; fluorescein isothiocyanate (FITC)-labelled concanavilin A (ConA), FITC-labelled *Datura stramonium* agglutinin (DSA), and FITClabelled *Lycopersicon esculentum* agglutinin (LEA) (all from Sigma, St Louis). All FITClabelled lectins were applied as 20 μg/mL solutions in Tris-buffered saline (TBS) for 60 min, followed by 3 × 5 min washes in TBS. Samples were examined using appropriate narrow pass filters on an Olympus BX61 fluorescence microscope.

To examine the distribution of adhesiveness on tubules, individual freshly expelled tubules after draining (see above) were transferred to a wash solution in a plastic trough which contained a suspension of 0.5% w/v Bio-Gel P2 (45-90 m particle size) in 3.5% w/v NaCl, 10 mM sodium phosphate, pH 7.6. After 5 sec immersion, the tubules were washed 3 times in 3.5% NaCl, 10 mM sodium phosphate, pH 7.6 and were then drained and placed onto glass slides. After air drying, the tubules were examined by microscopy.

For scanning electron microscopy (SEM) expelled tubules were examined using a Philips XL30 FESEM microscope at an accelerating voltage of 2 kV.

### **2.4 Gel electrophoresis analysis**

Freshly expelled and drained tubules were allowed to adhere to a glass plate and were air dried. The tubules on glass plates were removed by peeling, leaving the layer of adhesive, and potentially other components of the tubule wall as a print on the glass (DeMoor et al., 2003). This material was collected by removal with a sharp razor blade and was then extracted in electrophoresis sample buffer, containing 2-mercaptoethanol. SDSpolyacrylamide gel electrophoresis (SDS-PAGE) was based on the method of Laemmli (1970) using Invitrogen NuPAGE Novex 4-14% Bis-Tris Gel with MES running gel buffer, at 180V for 60 min. Molecular weights were determined by comparison to globular protein standards (BioRad) using BioRad Quantity One v.4.4.0 software. For protein identification, gels were stained by Coomassie Blue R-250. Samples that had not been dried completely, but only sufficient to remove excess liquid, appeared to give samples that contained less insoluble material, although the yield of adhesive proteins was less.
