H-bonded OH

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

0.04

2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000Wavenumber (cm-1)

Re(

D)

or Im

'(D)

Amide N-H

CH2, CH3

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

0.04

0.045

0.05

800 1000 1200 1400 1600 1800 2000

Wavenumber (cm-1)

Re(

D)

or Im

'(D)

Amide I

C=O

Amide II CH2, CH3

C-O

Figure 2. Re D ( ) and Im'D ( ) spectra for (a) the upper range of wavenumbers and (b) the lower range of wavenumbers.

41.5

42

42.5

43

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44

44.5

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Wavenumber (cm-1)

(degre

es)

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Wavenumber (cm-1)

(degre

es)

(a)

41.5

42

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Wavenumber (cm-1)

(degre

es)

140

145

150

155

160

165

170

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Wavenumber (cm-1)

(degre

es)

(b)

Figure 3. Simulations to illustrate the sensitivity of and to changes in film thickness and in water content. (a) Simulations of spectra for a film containing 39 vol.% water and having two different thicknesses: 39 nm (—) and 59 nm (—); (b) Simulations of spectra for a 49 nm thick film containing two different water contents: 29 vol.% (—) and 49 vol.% (—).

41.5

42

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43

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44

44.5

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Wavenumber (cm-1)

(degre

es)

140

145

150

155

160

165

170

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Wavenumber (cm-1)

(degre

es)

Figure 4. Ellipsometry spectra ( (top) and (bottom)) obtained from a mucus film in an atmosphere with RH = 74.3%. The black line is the best fit to the data using an EMA model for a 49 nm thick film containing 39 vol.% water.

0

10

20

30

40

50

60

0 20 40 60 80 100

Relative Humidity (%)

Wate

r Conte

nt

of Film

(vol.

%)

Figure 5. Volume fraction of water in the mucus film as a function of the RH of the atmosphere. The line is a prediction based on the Flory-Huggins expression using a best-fit value for the polymer/solvent interaction parameter of = 0.54.

Hydrophilicity of Pedal Mucus Trails of the Giant African Land Snail

Ben Lincoln, Tim Simpson, and Joseph KeddieDepartment of Physics, University of Surrey, Guildford, Surrey GU2 7XH, England

Giant African land snail (image courtesy of Annette

Goodman)

IntroductionMucus Trails Gastropods, such as slugs and snails, secrete a trail of mucus (i.e. a "snail trail") from their pedal gland while travelling across a surface [1]. The unique mechanical properties of snail pedal mucus enable the animal's locomotion while also causing the mucus to function as an adhesive to the substrate [2].

The mucus trail performs a number of other functions [1], including the provision of mechanisms for re-tracing a path (i.e. "homing") and for finding a mate of the same species by following a trail [3].

An understanding of the functionality of trail mucus, including its interactions with water vapour, can therefore lead to a means of controlling the reproduction of snails and thereby limiting their impact on the environment, especially vegetable crops [4].

It has been reported [5] that the presence of snail pedal mucus on a surface influences the settlement of other adhering marine organisms (such as barnacles) to that surface. The hydrophilicity or hydrophobicity of a substrate, in particular, has been found to influence the settling of other organisms [5], which suggests a reason why a mucus trail will have an impact.

The main constituent of gastropod mucus is a complex of proteins and polysaccharides. This complex is usually classified into the broad categories of mucopolysaccharides and glycoproteins.

Previous research characterising the "wettability" of mucus has only been semi-quantitative. Specifically, relative comparisons of hydrophobicity of pedal mucus trails were made via contact-angle measurements [5].

Infrared spectroscopic ellipsometry (IRSE) Infrared spectroscopic ellipsometry (IRSE) is a non-invasive technique that can determine the structure and optical constants of thin films and bulk materials.

IRSE has been shown to be a quantitative tool for the measurement of the concentration of biological molecules in a thin layer [6] and for the determination of the structure of organic films [7].

Objectives• To develop IRSE as a technique to detect snail mucus trails in their native state on a solid substrate. • To characterise the hydrophilicity of snail pedal mucus by determining the equilibrium water vapor sorption at constant temperature as a function of the relative humidity.

Conclusions Infrared ellipsometry has been used to identify the presence of mucus trails (about 30 nm thick) deposited by the giant African land snail (Achatina marginata) onto a solid substrate.

Characteristic chemical groups for gastropod mucus, including CH2, CH3, CO, C=O, and - most importantly - the amide I and II bands, are identified.

The equilibrium water vapour sorption isotherm of the mucus trail can be described using a solution model with a Flory-Huggins polymer/solvent interaction parameter of = 0.54 0.1. This value is similar to what is obtained for very hydrophilic synthetic polymers, such as poly(vinyl pyrrolidone).

The mucus trail remains hydrated under typical ambient conditions. The observed water sorption is much greater than what has been found in simple proteins (e.g. lysozyme and bovine serum albumin).

The hydrophilicity of the mucus might influence the trailing and homing mechanisms of snails, and it might also provide anti-bacterial protection for the snail.

IR spectroscopic ellipsometer

Discussion of Results There is an extensive literature on the measurement of water vapour sorption in poly(amino acids), peptides and proteins, as has been reviewed elsewhere [8]. There is a qualitative similarity between the water vapour sorption of synthetic glassy polymers (such as poly(vinyl pyrrolidone) and that of biological macromolecules [8]. Accordingly, the thermodynamic description of solvent sorption that has been developed for synthetic polymers can, as a first approximation, be applied to natural macromolecules.

When a polymer is in equilibrium with water in an atmosphere with an activity a, the volume fraction of water, fw, sorbed in the polymer can be found from

where is the Flory-Huggins polymer-solvent interaction parameter and N represents the number of water molecules that are required to equal the volume of the polymer molecule.

The parameter is a gauge of the interaction energy between the polymer and water at a molecular level. The higher its value, the more hydrophobic is the polymer. A value of = 0.5 represents a theta-solvent.

Measurements of water sorption in the same mucus film as a function of RH were used to characterise hydrophilicity, using as a gauge for comparison to other substances.

Water uptake increases strongly when RH increases above 60%. When RH = 84.3%, the volume fraction of water increases to 53%. This observed trend was reproduced in other experiments.

A value of 0.54 0.1 is obtained from the best fit, indicating that the mucus is very hydrophilic.

2)1(11

1lnlnwww N

a

UniS

84

85

86

87

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Wavenumber (cm-1)

Ψ

280

290

300

310

320

500 1000 1500 2000 2500 3000 3500 4000

Wavenumber (cm-1)

Δ (Degrees)

Figure 1. IR ellipsometry spectra (both (top) and (bottom)) obtained from a bare gold-coated substrate ( ) and from a partially-dried mucus film in the ambient atmosphere on the same substrate ( ).

(

deg

rees)

(d

eg

rees)

Key Equations for Ellipsometry Analysis:

Ellipticity, :where is related to the change in light’s amplitude upon reflection, and is related to the change in phase.

Optical Density, D:where the “o” superscript refers to the original surface, and “f” refers to the final (or altered) surface. New groups on a surface lead to a peak in Re D and a step with a negative slope in Im D.

ietan

f

oD ln

References1. M. Denny in The Mollusca, Vol. 1: Metabolic Biochemistry and Molecular Biomechanics, P.W. Hochachka, ed., Academic Press: London (1983), Ch. 10, pp. 431-437.2. M. Denny, Nature, 285 (1980) 160.3. T.J. Trott and R.V. Dimock, Mar. Behav. Physiol., 5 (1978) 91.4. J.M. Cottrell, I.F. Henderson, J.A Pickett, D.J. Wright, Comp. Biochem. Physiol. B Biochem. Molec. Biol., 104 (1993) 455.5. S.P. Holmes, A. Cherrill, and M.S. Davies, J. Marine Biol. Assoc. U.K., 82 (2002) 131.6. E. Garcia-Caurel, B. Drévillon, A. De Martino, and L. Schwartz, Appl. Opt., 41 (2002) 7339.7. D. Tsankov, K. Hinrichs, A. Röseler, and E.H. Korte, Phys. Stat. Sol. A, 188 (2001) 1319.8. S.L. Shamblin, B.C. Hancock, G. Zografi, Eur. J. Pharmaceutics Biopharm., 45 (1998) 239.