Probing the compositional and rheological properties of gastropod locomotive mucus

Most natural materials remain inside or close to the body, allowing for repair and reconfiguration. However, some species have evolved the ability to produce and use materials outside their bodies. Termed ecto-secretions, these are a remarkably overlooked yet important class of materials that are selected to perform in extreme environments and facilitate a range of biological functions, from structural (silks) to chemical (venoms) (Casewell et al., 2013; Flórez et al., 2015; Avella et al., 2021). Gastropod mucus is a prime example of such an ecto-secretion that when excreted as a thin layer (10–20 µm) aids locomotion, adhesion, and defence; prevents desiccation and infection; and in some cases, even serves as a substrate for microbial “farming” (Chase et al., 1980; Luchtel et al., 1991; Peck et al., 1993; Kim et al., 1996; Perez-vilar and Hill, 1999; Smith and Morin, 2002; Thornton, 2004; Artacho and Nespolo, 2009; Lai et al., 2009; He et al., 2016; Dhanisha et al., 2018). However, despite its natural ubiquity and utility, only a handful of studies have specifically focused on gastropod mucus alone (Denny, 1980; Denny, 1984; Denny and Gosline, 1980; Bretz and Dimock, 1983; Deyrup-Olsen et al., 1983; Hawkins, 1992; Cottrell et al., 1994; Davies and Hutchinson, 1995; Davies and Hatcher, 1999; Smith et al., 1999, Smith et al., 2009; Skingsley et al., 2000; Smith and Morin, 2002; Struthers et al., 2002; Pawlicki et al., 2004; Ewoldt et al., 2007; Werneke et al., 2007; Ewoldt et al., 2009; Braun et al., 2013; Newar and Ghatak, 2015; Zhong et al., 2018; Fung, Gallego Lazo, and Smith, 2019; O’Hanlon et al., 2019). This limited amount of knowledge surrounding the composition and structure of gastropod mucus is further compounded when considering its material and mechanical properties.

Although gastropods have captivated researchers for centuries, it was not until the 1970s that Denny (1973) and Denny (1984) systematised the study of gastropod mucus with a broader vision and through a combination of experimental and theoretical studies related a range of mucus’ physical properties to the animal’s biology and habitat (Denny, 1984). Denny was the first to study the mechanical (rheological) properties of gastropod locomotive mucus, demonstrating that in the slug Ariolimax columbianus, its shear stiffness is indirectly proportional to its water content (degree of hydration) (Denny, 1984). He proposed that the mucus polymers in gastropod locomotive mucus contributed to these properties by responding to their degree of hydration. For example, as the water content of mucus is reduced, the preferential interactions of the mucus polymers with water begin to switch to interacting with other mucus polymers, increasing the number of intermolecular associations and, therefore, stiffness.

Twenty years after Denny’s studies, Ewoldt et al. (2007) extended this line of research and compared the non-linear rheological properties of locomotive mucus from a snail (Helix aspersa) and a slug (Limax maximus). Their significant findings suggested that the timescale by which mucus is deformed determines whether it behaves as an adhesive or a lubricant. Using a novel rheological fingerprinting technique, they categorised mucus as having viscoelastic properties and behaving as a non-Newtonian gel (Ewoldt et al., 2007). More recently, Fung, Gallego Lazo, and Smith (2019) attributed the rheological properties of Arion subfuscus adhesive mucus to a double network of protein chains with sacrificial bonds and carbohydrates interacting with metal ions, both of which can readily reform if broken.

In addition to the degree of hydration or concentration, from a polymer science perspective, molecular weight (i.e., polymer chain length) could be equally, if not more, influential in determining flow properties (Ferry, 1980). However, this link has been somewhat overlooked to date. Most mucus compositional studies have tended to use the simplicity of SDS-PAGE to identify the molecular weight of proteins in gastropod mucus, for example, characterising the marine snails Lottia limatula and Haliotis diversicolor; terrestrial slugs Arion subfuscus and Arion ater; and the garden snail Helix aspersa (Cottrell et al., 1994; Smith et al., 1999, Smith et al., 2009; Smith and Morin, 2002; Pawlicki et al., 2004; Ewoldt et al., 2007; Werneke et al., 2007; Guo et al., 2009; Wilks et al., 2015). However, more specialised mass spectroscopy and chromatography have also been used for determining the molecular weight of components in the marine snails Patella vulgata and Dendropoma maxima; terrestrial slug A. subfuscus; and terrestrial snails H. aspersa, Eobania vermiculata, Thebe pisana, and Monacha obstructa (Davies and Hatcher, 1999; Pawlicki et al., 2004; Werneke et al., 2007; Smith et al., 2009; Sallam and El-Wakeil, 2012; Klöppel et al., 2013). However, the link between mucus polymer morphology and its influence on mucus performance remains to be determined.

Hence, a cohesive understanding, within an appropriate evolutionary context, between mucus’ molecular components and flow behaviour across a range of species is currently missing. Therefore, to differentiate between the factors that could influence mucus rheology, we propose using UV-vis spectroscopy to determine protein concentration or hydration level between samples and SDS-PAGE to identify proteins and their molecular weight. The approach proposed here has not been explored previously, as most studies incorporating SDS-PAGE with UV-vis tend to focus on a specific protein of interest, not probing the entire composition of mucus. In addition, to the authors’ knowledge, something as seemingly trivial as mucus protein concentration has surprisingly not been reported.

Our hypothesis is that mucus polymers influence the mucus phenotype, and in line with our wider classification of these materials as ecto-secretions, mucus proteins will be a key component in helping deliver functionality for the required timescale of use by the animal. Hence, this work presents an initial foray into this area through a combination of thermal (thermogravimetric analysis (TGA) and rheological ramp temperature tests), compositional (UV-vis and SDS-PAGE), and functional (rheology) techniques to characterise and compare locomotive mucus across six different terrestrial gastropod species: Achatina fulica (Lissachatina fulica), Cornu aspersum, Cepaea nemoralis, Arion ater, Arion hortensis, and Limax flavus.