The tenacity of the limpet, Patella vulgata L.: An experimental approach

217

.I. exp. mar. Biol. Ecol., 1981, Vol. 54, pp. 277-308 Elsevier,‘North-Holland Biomedical Press

THE TENACITY

OF THE LIMPET,

AN EXPERIMENTAL

J.-F. GRENON’ N.E.R.C.

Unit of Marine

Invertebrate

PATELLA

VULGATA

L.:

APPROACH

and G. WALKER

Biology. Marine Science North Wales, U.K.

Luboratorirs,

Menai

Bridge,

Anglesey,

Abstract: It has been shown that adhesion of the limpet, PateHa vulgata L. is influenced by both physical and physiological factors. The tenacity is sensitive to surface properties of the substratum, varying inversely with the contact angle which water makes with a substratum. This can be explained in terms of thermodynamics. Surface roughness also affects tenacity and this is explained in the same manner. Different angles of detachment were tested and it was clearly shown that when a strong peeling component was introduced, a much reduced force was needed to detach a limpet. Contrary to a normal pull. when a shear pull is exerted the force is not proportional to the surface area of the foot. It has also been shown that the speed of separation affects the measured tenacity; there is a speed at which tenacity will be maximum. The effect of water temperature on tenacity has been tested, tenacity increasing with rising temperature (7, 13, 20°C). At the higher temperatures limpets are able to contract the foot muscles more powerfully, indicating that increased foot rigidity increases tenacity. By measuring the tenacity of limpets left out of water for different periods of time it has been shown that desiccation has no effect on tenacity, but a change from aquatic to aerial respiration increases tenacity. Tenacity has also been measured when the limpets have been subjected to a reduction in metabolic rate. The effect of both anoxia and narcotization shows that reduced muscle tonus, especially in the foot. results in decreased tenacity. These results further demonstrate that foot rigidity is essential for efficient adhesion. Limpets from different habitats (exposed and sheltered) and vertical distribution (high and low level on shore) exhibited no differences in tenacity. During locomotion limpets leave a mucous trail, most of the mucus being confined to the edge of the trail. Water is incorporated anteriorly under each new locomotory wave and these pockets of water are used to release the mucus from the substratum during locomotion. It is concluded from this study that limpet adhesion can be explained solely by the tackiness of the pedal mucus, tack being due to the stored elastic energy within the mucous layer itself.

INTRODUCTION

As for many other invertebrates, marine gastropods are able to attach to substrata in order to maintain their position against environmental forces such as waves, wind, current and gravity; adhesion to a substratum will also help in protecting against predation. Amongst these gastropods, limpets are probably the most successful group in terms of their adhesive power. The mechanism by which limpets adhere to substrata has intrigued workers for a long period of time and the various theories proposed are still able to stimulate controversy. A summary of the earlier studies on limpet adhesion together with the various proposed adhesion mechanisms ’ Presitnt address: Canada.

G.I.R.O.Q.,

0022-0981!81/0000-OOOO/$

DCpartement

de Biologie,

02.50 0 Elsevier/North-Holland

UniversitC

Biomedical

Laval,

Press

QuCbec, P.Q. GlK

7P4,

J.-F. GRENON

278

is given in Table of adhesion, atmospheric of limpets

I. Suction

but suction pressure may

AND

G. WALKER

has been proposed

a number

cannot

(1.033 kg ‘cm-‘).

be much

higher

Summary

than

It is now well established 1.033 kg .crn-’

Tenacity (kg .crn-‘)

1711

Woodward, 1875 Aubin, 1892 Davis, 1895

Pat& vulgata Patella vulguru Ptrtdlu w&uta

1.03 3.82

Pieron,

Porrlla vulguta

1.40

Menke, 1911 Parker, 1911 Wells, 1917 Loppens. 1922 Abe, 1931 Thomas, 1948

Patella Putella Acmuea Patella Acmaea Cellunu

3.50

Crisp,

Patella sp

1909

1973

Miller,

1974

Branch

& Marsh,

1978

sp. sp. scubtx vulgutu dorsuosu trumosrricu

Acmueu prltu Acmaea scutum Patellu cochleur P. urgenvillei P. longicostu P. grunuluri.c P. ~yranulinrr P. odus

“ 15 kg for adult limpet. ’ 4 to 5 x atmospheric pressure,

of times as the sole means

forces per unit area (tenacity) (Table

greater

than

that the tenacity I). Other

workers

of the earlier studies on limpet adhesion.

Species

Author Reaumur,

explain

Proposed adhesion mechanism Secretion of an adhesive substance Suction “Adhesion” of two surfaces closely applied together Suction and interlocking of the foot with the substratum Thin film of tacky mucus Suction

3.94 ,.a93 b

0.67 0.88 5.18 4.67 4.40 3.25 2.17 1.95

Suction Suction mucous adhesion Tackiness of viscous pedal mucus Tackiness of pedal mucus : Stefan adhesion

Mucous cohesion tension

and surface

i.e.: 4.12 to 5.15 kg ‘cm -?.

believed that the high tenacity of limpets was due to the tackiness of the pedal mucus. Crisp (1973) has recently proposed that limpets adhere by tacky adhesion as described by Stefan (1874). Over the years different interpretations for the term tack have been used. A general definition of tack is given by De Bruyne (1944) where a tacky adhesive is defined as an “adhesive in a mobile state which can resist forces tending to cause relative motion of the surfaces with which it is in contact”. Bikerman (1947) and Erb & Hanson (1960) have also defined tack in a similar way. Banks & Mill (1953) however, introduced a more specific definition of tack : “forces or energies involved

OF P.4TELLA

TENACITY

in the separation viscous tackiness.

of two surfaces

liquid”.

Following

Wetzel

their definitions.

in general

this definition

(1957) and Wake

closely only

separated

highly

by a film of highly

viscous

(1965) later introduced

Wake (1965) defined

279

C’c’LG.4 T.4

liquids

the notion

tack as follows : “properties

can cause of time in

of an adhesive

substance whereby momentary contact of the substance with a solid is followed immediately by a resistance to any attempted separation”. As pointed out by Hoekstra & Fritzins (1951) tackiness is a complex property, a combination of a number of different simple properties such as adhesion, cohesion, surface tension, viscosity, yield value, etc., but is not the sum of these. Tack can be more easily understood by examining the mathematical equations which govern it.

TACK:MATHEMATICALCONSIDERATIONS

The earliest work on tack was carried out by Stefan (1874) who studied the problem using both a mathematical and experimental approach. He measured the time involved in the process of separating two circular flat discs of radius R immersed in a liquid of Newtonian viscosity n at a constant force F. His findings are given by the following equation :

(1) where h, and h, are the initial Rearranging

equation

and final separation

distances,

respectively.

(1) we obtain : F=3mR4 4t

1 h; C---J

1 h;

’

(2)

where F is the force necessary to separate the two discs from h,) to h, in time t. If /I, % h,), however, equation (2) becomes: F = 3mR4 4th’

.

In the Stefan approach, viscosity is the tack determining factor and the force is merely produced by the viscous flow of the liquid towards the centre of the discs. The Stefan equation is, in practice, more conveniently expressed in a differential form (see Banks & Mill, 1953; Crisp, 1973; Grenon et ul., 1979): F = 3mR4 2h3

dh dt ’

(4)

This is the Stefan equation for separating two discs at a constant velocity dlzldt. The force is inversely proportional to the cube of the separation thickness of liquid.

280

J.-F. GRENON

AND G. WALKER

A similar transformation can also be performed on the original equation given by Reynolds (1886) in which elliptical plates were considered :

(5) where a and c are the semi-axes of the elliptical plate and t, n, F and h,, h, have the same signification as in the Stefan equation. The differential form of this equation is given by: F=

3zna’c’

dh (a2 + c’)hi dt .

16)

The Stefan equation (2) is given for two discs completely immersed in a Newtonian liquid. Healey (1926) derived the following expression in the case of two discs separated by a drop of Newtonian liquid : F= 3nV2 -38nt

1 7ipTy

1 ) ’

where V is the volume of the drop. If h, $ h, and if the liquid initially fills the space between the discs so that V = nR”h, then equation (7) becomes: F =i 3nnR4 8th? . The force required to pull two discs separated by a drop of Newtonian liquid is, therefore, half the force necessary to separate the discs when they are fully immersed. The latter equation only applies to separations normal to the surfaces. However, Crisp (1973) compared this force with that needed to slide the discs sideways and gave the following equation : F = ItR’n dh 7-x

When the discs are separated by a liquid of non-Newtonian viscosity, account should be taken of the variation of shear stress with the rate of shear. The rate of shear (dv/dq’) is related to the shear stress (r) by the following equation for liquids with a yield point:

where J’is the yield value, x the mobility of the liquid and n a numerical constant (Houwink, 1937). Scott (1931) derived the following expression for liquids with a yield point (i.e. e is finite) and a value of n = 1:

TENACITY

where FL is the limiting volume

force beyond

of the liquid between

When

OF PATELLA

which

2x1

VULGAT.4

no separation

the two discs and h the thickness

two discs are separated

by a drop

distance is small, surface tension becomes to separate the two discs is given by: F I

of liquid

appreciable

=2vT h”

and

will take place,

v the

of liquid. the initial

and the force

separation (4)

needed

(12)

where V is the volume of the drop, T the surface tension and h the thickness of liquid (Poynting & Thomson, 1934). If the force applied is F,, the effect of surface tension will be negligible. Stefan-type of adhesion presupposes a perfect wetting of the adherend and as the two discs separate a single filament will be drawn towards their centres by viscous flow. Exceptions occur with complex liquids, however, and they have been classified as “nonStefan” or “ultra-Stefan” (Banks & Mill, 1953; Erb & Hanson, 1960). In these instances a multi-filament system develops as the discs separate, as a result of cavitation within the adhesive layer (Banks & Mill, 1953) or due to the elastic behaviour of the adhesive (Voet & Geffken, 1951). In these two situations the force of adhesion is smaller than predicted by the Stefan equation (Equation 2). Banks & Mill (1953) derived the following modification of the Stefan equation:

A - P, =

$$ ,

(13)

where A is the pressure on the liquid at the outside of the discs (usually equal to 1 atm.), PO is the pressure at the centre of the liquid and U is the velocity of separation. In this equation, the pressure at the centre of the liquid (P,,) is related to the separation velocity (v>. Thus, the maximum stress which the liquid can sustain gives a maximum limit to the velocity of cavitation-free disc separation. When cavitation occurs, the Stefan equation is no longer valid to relate force and separation

rate (Erb & Hanson,

1960).

Voet & Geffken (1951) considered tack basically as a viscoelastic reaction to stress. The theory of viscoelasticity of Maxwell (1867) is the fundamental idea underlying their views and can be diagrammatically represented by a spring and a dashpot connected in series. When a viscoelastic material of the Maxwell type is subjected to stress the material deforms due to the extention of the spring. If the spring is stretched, the energy stored in the spring will relax due to the flow of the dashpot. The time needed to relax the stored energy is known as the Maxwell relaxation time and is.the ratio of viscosity to elastic modulus. If the duration of the stress is much longer than the relaxation time, the liquid will flow and if it is smaller, the liquid will react in an elastic fashion. If the duration of the stress is of the same order of magnitude as the relaxation time, a truly viscoelastic response will be observed.

J.-F. GRENON AND G. WALKER

‘X2

It has been shown Putella

previously

(Grenon

vulgata is a viscoelastic

material.

& Walker,

1980) that the pedal mucus

Measurements

of tenacity

under different conditions are necessary, however, to determine have an effect on the adhesion mechanism. Such measurements the present

of

of P. vufgata

those factors which form the basis of

study.

MATERIALS AND

METHODS

Different sized specimens of Patella vulgata L. were collected from various shores of the Isle of Anglesey, North Wales. Great care was taken during collection not to damage the animals. Any damaged limpets were discarded. The limpets were brought into the laboratory and placed in tanks of running sea water (maintained at 13 “C, except for the experiment on the effect of temperature) containing the experimental substrata (see p. 295) to which the animals were allowed to adhere. MEASUREMENT OF THE FORCE OF ADHESION The shells of attached limpets were carefully cleaned and stainless steel hooks glued to the shells with epoxy adhesive (Araldite, Ciba-Geigy) so that each hook was close to the central axis of the animal (see Grenon et al., 1979). The force needed to detach an animal was measured with an apparatus already described (Grenon et al., 1979). In order to standardise measurements, just prior to the detachment, the shell of each limpet was tapped so inducing the animals to clamp down firmly onto the substratum. MEASUREMENT OF FOOT SURFACE AREA The surface

area of the foot of limpets

attached

to transparent

substrata

(glass

and Perspex) was measured from photographs taken from below just before commencing the force measurement. For limpets attached to non-transparent substrata (e.g. slate) the foot surface area (A) was determined

using one of the following A = 2.222SL + 0.238SW

(R = 0.9723, n = 55, P < O.OOOl), for limpets and a shell width (SW) > 1.62 cm, and:

relationships: - 3.774

with a shell length

A = 0.72OSL + 0.427SW

(SL)

> 1.86 cm

- 0.944

(R = 0.9380, n = 37, P < 0.0001) for limpets with a shell length < 1.86 cm and a shell width < 1.62 cm. Although these limits have been chosen arbitrarily, they allow determination of foot area with more precision, especially in the lower size range. These relationships were established from the photographic data collected

TENACITY OF PATELLA as above

using

measured

with micrometer

multiple

regression calipers.

analysis.

283

VULGATA

The

shell

length

All the data were processed

and using

width

were

a DEC-10

computer. CINEPHOTOGRAPHY AND VIDEOTAPE RECORDINGS A sequential analysis of limpet detachment (from below and sideways) was carried out using a Bolex 16RV cin& camera. The filming speed was 64 frames . s- ’ The videotape recordings were carried out with a time lapse videotape recorder (Model SV-61 2E(K), Hitachi Electronics Ltd.) coupled to an EMI Surveyor II camera. DEFINITION OF TERMS USED IN THIS STUDY Normal force: force measured by applying a pull normal to the substratum, i.e. at right angles. Shear force: force of adhesion measured with a pull parallel to the substratum. Peel force: force of adhesion measured with a pull at an angle with the substratum ( # 90”, # OO). Tenacity: force of adhesion per unit of foot surface area. Normal tenacity : tenacity calculated from a normal force. Peel tenacity: tenacity calculated from a peel force. Shear tenacity: tenacity calculated from a shear force. Separation force: force of adhesion needed to detach a limpet from the substratum.

RESULTS AND

DISCUSSION

PHOTOGRAPHIC ANALYSIS OF LIMPET DETACHMENT Cinephotography of the detachment of limpets showed that the shell is lifted some distance clear detachment. The distance moved depends on the the animal and also the amount of contraction of

pulled normal to the substratum of the substratum before actual speed of detachment, the size of the foot muscles. When the shell

was raised in this manner, it was observed that both the foot length and width were reduced sometimes by up to 10 or lS”/,. This means that the area of the foot in contact with the substratum can be reduced by 20 to 30% before the limpet actually becomes detached. As the shell is lifted the pull is transmitted to the dorsoventral muscles and because of their transverse insertion on the foot sole epithelium (dorsoventral muscles becoming transverse) the edge of the foot is drawn in. The actual detachment process followed a regular pattern. The centre of the foot is released first, the edge of the foot being the last area to leave the substratum. As soon as the foot was off the substratum, the region around the edge curled inwards. Even if the hook is very carefully centred in order to lie in the pulling axis,

J.-F. GRENON

284

however,

a small

peeling

component

not affect the forces measured EFFECT

effect

OF DIFFERENT

AND G. WALKER

is sometimes

introduced

( < loo). This does

(see p. 289).

CONDITIONS

ON THE TENACITY

OF P. VULGATA

of substratum

Emersion. In nature, limpets are found on substrata not only of variable roughness but also of different surface properties. Adhesion of a liquid to a solid is extremely sensitive to surface properties, especially the wetting properties (Adam, 1941). The effect of different substrata on the normal tenacity of P. vulgata was investigated. The relationships between the force of adhesion and the surface area of the foot are shown in Fig. 1 for limpets attached to glass, rough slate, smooth slate, Perspex and Teflon. For the last substratum, Perspex plates coated with polytetrafluoroethylene (P.T.F.E., Fisons Scientific Apparatus, Loughborough, England) were used. Limpets were taken out of water just before the force measurement. The experiment was carried out at room temperature (20 f 1 “C) and the pulling speed was 0.10 cm .s-‘. The equations of regression for the best tit curves are given in Table II. The equations of regression are all highly signilicant. The

TABLE II Equations of regression for the best tit curves of the relationship between the force of adhesion (F) and the surface area of the foot (A) for different substrata under emersion conditions: Y, coefficient of correlation; P, probability of r: n, number of observations. Substratum Glass Rough slate Smooth slate Perspex Teflon

Equation F = F= F = F = F =

of regression

0.673 1.531 I.928 1.945 0.758

A ‘.672 A’.‘46 A - 0.304 Ao.875 A”.“”

r 0.9240 0.9246 0.8549 0.8466 0.8057

P < < i < <

0.001 0.001 0.001 0.001 0.001

n 15 47 19 43 23

relationship between the force of adhesion and the surface area of the foot is slightly curvilinear for all substrata except for smooth slate, where a linear regression gave the best tit. As the area of the foot of animals attached to non-transparent substrata had to be estimated using multiple regression analysis of the foot area against the shell length and shell width, it is possible that a small error may have been introduced leading to the curvilinear relationships. It is also possible that if the number of observations was increased, the relationships would have been linear. It should be noted that linear regression was also highly significant (P < 0.0001). Furthermore, the curvilinearity only becomes appreciable at very large foot surface areas, areas which are not found in nature. Also it is evident that when the area of

TENACITY OF PATELLA

285

VULGATA

4

2 AREA

OF

AREA

FOOT, cm2

AREA

OF

6

IO

---z-------

OF FOOT, Cd

FOOT, cm2

30 I

, “_?___._

_~__.__ 2 AREA

4 OF

6

8

FOOT Cd

Fig. 1. a--e, relationship between the force of adhesion and the foot surface area of Putelkt wlgurcr attached to glass, rough slate, smooth slate, Perspex and Teflon respectively: limpets were detached in air at room temperature (20 f I “C) at a pulling speed of 0.10 cm ‘s - ’ ; best fit curve and linear regression line forced through the origin are both given; f, comparison between the five linear regression lines forced through the origin.

J.-F. GRENON

286

AND

G. WALKER

the foot is zero, the force must also be zero. These considerations therefore allow linear regression to be forced through the origin. The equation will then take the form of:

F=KA, where

F and A are the force of adhesion

and foot surface area respectively,

K being

the equation constant (slope) (kg .crn-I). The use of the K values will allow comparisons between the different substrata to be standardised. The K values for animals attached to the various substrata are given in Table III. The contact angles

TABLE III Tenacity

(K values k SE) of Prrtelh vul~c~tcrand contact Substratum

Contact

angle of water on different Tenacity

angle

(kg

substrata.

cm -‘) + SF

(“) Glass Rough slate Smooth slate Perspex Teflon

2.320 2.284 1.857 1.649 0.785

33 38 51 65 92

i * + + +

0.192 0.208 0.121 0.188 0.218

for water (measured photographically) on these substrata are also given in order to show the different surface properties. The regressions through the origin for the different substrata are shown in Fig. If. The comparison between the K values has been accomplished using the Newman-Keuls multiple range test (SNK test) (Zar, 1974) and the q-values are given in Table IV. The SNK test shows that there is no significant difference (P = 0.05) between the tenacity of P. vulgata attached to glass and rough slate and between smooth slate and Perspex. The others are significantly different (P < 0.05). Although no significant difference was found between smooth slate and Perspex, it seems that for smooth surfaces, the tenacity varies inversely with the contact angle. This is in agreement with the thermodynamic equation of Young-Dupre for the work of adhesion of a liquid wetting a solid : WSL

=

YL,,(l +

cosq 1

(14)

where Ws, is the work necessary to separate the liquid from the solid (work of adhesion), yLA is the surface tension at the liquid-air interface and 8 the contact angle made by the liquid (see Tabor, 1951). Equation (14) shows that the work of adhesion of a liquid to a solid is inversely proportional to the contact angle. A small contact angle implies a better ability by a liquid to spread. If limpets become attached to a wettable surface (small contact angle), they will be able to spread a thin mucous layer evenly between the foot and the substratum. Furthermore, the

TENACITY

OF P,4 I-ELLA

I’ULG.4 Td

2x7

molecular forces are greater at smaller contact angles and this explains the negative correlation between the contact angle and tenacity. Tal31.r IV q values calculated

for comparison of the tenacity (SNK test) obtained for limpets attached substrata : *. significant at P <:0.05; **. not significant. Glass

Rough

slate

Rough

slate

3.785*

Perspex

4.903%

6.443*

Teflon

9.230*

10.980*

slate

slate

Perspex

0.2x3** 3.140*

Smooth

Smooth

to different

I .678** 6.870*

5.937*

The effect of substratum roughness on the tenacity of P. vufgata can be appreciated by comparing the K values obtained on smooth and rough slate. The SNK test (Table IV) shows that the K value for rough slate is significantly greater than the K value for smooth slate. This can also be explained in thermodynanlic terms. Roughness of a surface has the effect of altering the contact angle: if smooth material gives a contact angle >90”, roughness increases this angle further, but if the contact angle is <90”, roughness decreases the angle (see Davies & Rideal, 1963; Zisman, 1962). Comparison of the contact angles on smooth and rough slate (Table III) shows that roughness decreases the contact angle (51’ to 38”). This experiment has shown that for the five substrata used the force of adhesion of P. vulgatn normal to the substratum is proportional to the surface area of the foot. In an early study, Menke (191 I) found no such relationship. Such a relationship has been found for Acnmeu sp. (Wells. 1917), T/rnis emavginata and ~cguf~z ,~~neb~u~i~~ (Miller, 1974), however, and for six other species of P~~te~ia:P. ~~~~~~e~~, P. ~~~~efl~~il~~~, P. ~on%ico.sta,P. ~run~~~a~i.s,P. ~~ra?li~t~~l~~, P. oc~i~.~ (Branch & Marsh, 1978). immeuion. The effect of substrata has also been investigated under immersion conditions. In these experiments the tenacity on three substrata, rough slate (hereafter called slate), Perspex and Teflon, was compared. The relationships between the force of adhesion and the foot surface area are given in Fig. 2. The experimental conditions were the same as those of the first experiment except that limpets were detached under water at a temperature of 13 “C. The equations of regression for the best Iit curves are given in Table V. A logarithmic relationship was found for sfate while for Perspex and Teflon a linear

J.-F. GRENON

288

relationship substrata.

AND

K values

gave the best fit. Again The K values (kg .cm-‘)

( f SE 0.040) for slate, Perspex

for comparison. thermodynamic

were used

are 2.447 ( f SE 0.119)

and Teflon,

the tenacities of limpets attached The three regression lines forced

G. WALKER

respectively.

to compare

the three

1.982 ( + SE 0.121), 0.974

The SNK test showed

to these three substrata are significantly through the origin are plotted together

A tension may occur at the mucus-water interface considerations discussed previously can be applied.

that

different. in Fig. 2d

and the same

b

25

1

5-

2

4

6

AREA

AREA

OF

OF

FOOT.

FOOT,

8

IO

cm2

cm2

AREA

OF

FOOT,cm2

AREA

OF

FOOT.

cm2

Fig. 2. a-c, relationship between the force of adhesion and the foot surface area of Pure//r/ rulgcrtcr attached to rough slate, Perspex and Teflon respectively: limpets were detached in water at 13 “C at a pulling speed of 0.10 cm .s- ’ , best fit curve and linear regression line are both given; d. comparison between the three linear regression lines forced through the origin.

TABLE

V

Equations of regression for the best tit curves of the relationship between the force of adhesion (F) and the surface area of the foot (A) for different substrata, under immersion conditions: r, coefficient of correlation; P, probability of r; n, number of observations. Substratum Slate (rough) Perspex Teflon

Equation

of regression

F= 1.949 A’ “’ F = 2.200 A - 1.049 F = 1.065 A - 0.804

r

P

n

0.9229 0.8613 0.9140


21 39 15

TENACITY OF PA TELLA YULGA TA

2x9

In most of the previous studies, tenacity of limpets has been measured out of water. Miller (i974), on the other hand, obtained all her data after pulling under water. No information exists, therefore, on the effect of immersion and emersion on the tenacity of limpets or any other gastropod. In order to test whether there is a difference between the force needed for detachment in water and out of water, data from the first two experiments were used. The Student t-test was used to compare the K values; tenacity was shown to be significantly higher in water for limpets attached to Perspex (t = 2.208, d.f. = 78, P < 0.05) but no significant difference between limpets in water and out of water on slate (f = 0.993, d.f. = 70) or Teflon (t = 1.680, d.f. = 34). It will be shown later, however, (see p. 297) that a longer period of emersion increases tenacity.

EJfect ofu peeling component. In the past, adhesion force measurements of gastropods have been performed using either a pull at right angles to the substratum (normal) or a pull parallel to the substratum (shear) and no information is available concerning the effect of pulling at other angles (peeling effect). In nature, predators of limpets such as birds and crabs are likely, in dislodging limpets, to introduce a strong peeling component. For this experiment, hooks were glued at the anterior or posterior part of the shell of animals attached to Perspex so that the hook lay parahel to the substratum, and the limpets dislodged with a pull normal to the subst~t~m (Fig. 3). The pullil~~

ANTERIOR

Fig. 3. Position

ofhook and

direction of

pull for shear and peel force measurements.

speed was 0.10 cm .s-’ and the experiment was performed out of water, at room temperature (20 Lt I YJ). The relationships between the force of adhesion and the foot surface area for front peel (front to back) and back peel (back to front) are highly signi~cant

J.-F. GRENON

290

AND G. WALKER

(Table VI). The front peel gives a slightly curvilinear relationship (Table Vi). In order to compare the direction of peel and the pull normal to the substratum (see first experiment, p. 284), the slope of the linear regressions forced through the TABLE VI Equations

of regression

Direction

of peel

of the force of adhesion different peels

E~~u~tio~l of repression

..__~_ Front Back

(!> and

F = 0.979 A ‘.liz F=

1.251 A -0.025

the surface

area

of the foot

(A) for two

: r. P, n, as before. i ..-___

.~_ 0.9713 0.8371

P _.-~-_ < 0.001
n 17 17

origin (iu values) were used. The K values (kg -cm-‘) are 1.237 (I SE 0.191) and 1.246 ( 3: SE 0.164) for front and back peel, respectively. The SNK test shows that there is no significant difference between front peel and normal pull (17= 4.45. P < 0.05) and between back peel and normal pull (q = 4.49, P < 0.05). The force needed to peel a limpet completely off the substratum is * 7.5’:;;of the normal force, so the difference is not excessively large. It was shown in an earlier study (Grenon & Walker, 1978) that a marginal gland secreting proteins and a marginal groove are situated around the anterior third of the foot. The fact that no difference was found between front and back peel tenacity indicates that the presence of the secretions of this gland and groove do not affect the peel tenacity. C~nephoto~~~phy showed that during peeling, limpets maintained a very rigid foot and a cohesive fracture initiated in the mucus was rapidly propagated. Cinephotography during detachment also showed that sometimes a small peeling component was introduced during pulls normal to the substratum (see p. 283). As a large peeling component shows reduction in tenacity of only 25”,,, there is no reason to believe that a small peeling component ( < 10’) has any significant effect on the value of the tenacities measured normal to the substratum. It was shown by Grenon & Walker (1980) that the rheological behaviour of the pedal mucus of P. vufgatu is more like a solid than a liquid (for stresses applied for a short time). This behaviour is reflected in the results obtained in the present experiment. i+~.! of’hori~ontal comrponenf ~shear). In the environment, forces acting parallel to the substratum (shear forces) are numerous (waves, currents, predators). Jones & Demetropoulos (1968) have measured horizontal forces generated by waves of the order of 1.25 kg . cm-? on exposed shores of Anglesey. To be able to measure the shear forces required to move them, limpets were allowed to attach to Perspex and the hooks fixed anteriorly as in the peeting experiment (Fig. 3). Perspex was preferred to slate because lower frictional forces

TENACITY

are generated detached

between

by pulling

OF PATELLA

the shell and the substratum. tangentially

291

VULGATA

to the surface

Limpets

at room

out of water

temperature

were

(20 + 1 “C)

and at a speed of 0.10 cm .s-‘.

91

I

8 7

6-

5-

r”

l-

.

.

-

.

.

. I

1

2

3

I

!

I

4

5 AREA

Fig. 4. Relationship between attached to Perspex: limpets

.

OF FOOT,

6

7

8

9

10

ctn2

the shear force of adhesion and the foot surface area of Pat& wlgatcr were detached in air at room temperature (20 + 1 “C) at a pulling speed of 0.10 cm .s-‘.

Fig. 4 shows that, for the data points obtained, no relationship between shear force and the surface area of the foot was found (Y = 0.3489, P = 0.116, n = 22). Shear forces as low as 0.95 kg for limpets with a foot area of 9.26 cm’ (0.102 kg .crn-‘) were recorded.

Although

there is no relationship,

shear forces are obviously

smaller

than the normal forces, the mean shear tenacity being 0.50 kg .crn-” (+ SE 0.11) which is 20-30% of the normal tenacity. Shear forces were also measured for animals attached to slate in order to evaluate the effect of the frictional forces. Only large limpets were used (foot surface area >4.5 cm’). The mean shear tenacity measured on this substratum was 1.75 + SE 0.23 kg .cm -’ (n = 22). This shows that E SOY,,of the measured tenacity was due to frictional forces. Videotape recordings incorporating a simultaneous view of the limpets being detached and the read-out from the recorder of the adhesion testing apparatus (see Grenon et al., 1979) indicating variation of shear force with time (F - t curve),

J.-F. GRENON

292

AND G. WALKER

were taken. A typical F - t curve is shown in Fig. 5. The first part of the curve (Fig. 5, A-B) corresponds to the shell being pulled forward, the foot remaining stationary. Then the curve becomes almost linear as the foot glides over the substratum but exerting some resistance (Fig. 5, B-C). This force reaches a maximum then declines. At the maximum point the foot is effectively detached from the substratum, i.e. the foot glides on the substratum but no resistance is recorded (Fig. 5, C-D).

I 2

1

/ 3

TIME, set

Fig. 5. Typical

curve showing

the variation

of shear force with time: see text for explanation

Miller (1974) has measured shear forces for a number of gastropod species (on Perspex, in water). She found that for Thais emarginata the shear force was 24% of the normal force. This is in agreement with the results obtained in the present experiment. Miller (1974) found a relationship during locomotion between shear force and surface area of the foot of Thais emarginata and two other species of Thais. Although she did not make any measurements of shear force on stationary animals, she presumed that the same relationship would hold. In the present experiment no relationship between the shear force and the foot surface area of stationary Patelta vz~igatacould be found. Warburton (1976) found a relationship between the shear force and the shell aperture area of stationary Patina pellucida and gave a K value of 0.256 kg .crnm2. In this latter case, limpets were settled on Laminaria digitata and the presence of mucilage on the algal fronds may have helped to reduce the friction of the shell and the foot against the substratum. The Stefan formula predicts that shear forces will be much less than the normal forces. The ratio of equation (9) and equation (4) gives:

TENACITY OF PA TELLA

293

VULGA T.4

so that for a limpet with a foot 3.5 cm in radius (assuming that the foot is a circle) and a pedal mucus thickness of 3 pm (Grenon & Walker, in prep.), the difference between the normal and shear pulls at the same separation speed woutd show that shear tenacity would be 4.89 x lO_“‘~ of the normal tenacity. Although these equations are given for Newtonian liquids these calculations in no way approach the actual data obtained in the present experiment. Even though Perspex has been used to reduce friction, it has not been eliminated completely. Friction plays an important role in situ preventing limpets being dislodged easily. Substratum roughness and the home scar produced by limpets will contribute by increasing frictional forces and creating mechanical interlocking so increasing shear tenacity.

Contrary to the various methods employed by previous workers, the apparatus used in this study (see Grenon et al., 1979) enabled limpets to be pulled at a constant speed and also the speed to be varied. This experiment was undertaken to see how the normal tenacity of Patella vulgata is affected by varying the pulling speed. Five different speeds were tested (0.03, 0.10, 1.00, 2.00 and 4.50 cm .s-‘) on two different substrata (Perspex and slate). Limpets were detached out of water, at room temperature (20 + I “C). The equation of regression for the best fit curves are given in Table VII together with the K values. The relationships between the force of adhesion and the surface area of the foot remains highly significant for the five experimental speeds on both substrata. In all cases, except for limpets attached to Perspex and detached at 0.03 cm ‘s”‘,

Equations of regression for the best fit curve of the relationship between the force of adhesion (t”) and the surface area of the foot (A) of Patetla vulgatu detached in air, at different pulling speeds: the K values of the linear regression forced through the origin are also shown (f SE); r, P, n, as before. Pulling speed (cm .s -I)

KkSE (kg .crn-‘)

Substratum

Equation of regression

r

0.03

Perspex Slate

F = 0.706 z4 + 0.968 F= 1.399/f’-“4

0.4394 0.8753

co.019
29 24

0.816 i 0.100 1.865 + 0.160

0.10

Perspex Slate

F= 1.945 A”.875 F= 1.531 ‘4’.‘46

0.8466 0.9246


43 47

1.649 i 0.093 2.284 & 0.103

1.00

Perspex Slate

F = 1.230 A ‘.“” F = 1.069 A ‘.“I

0.8640 0.8609


11 14

1.630 + 0.212 1.525 * 0.146

2.00

Perspex Slate

F = 0.591 A 1.379 F = 0.423 A ’ Iv2

0.9216 0.8943


26 14

1.123+0.072 0.616 + 0.052

Perspex Slate

F = 1‘132 ,‘to.s0() F = 1.566A tLe4s

0.6765 0.7375


20 26

0.712 &.0.092 0.885 + 0.070

4.50

P

I2

J.-F. GRENON

294

the best fit equation considered

linear

(P < O.Ol), except

AND G. WALKER

was the logarithmic

since

the exponent

for limpets

detached

form.

All these

of A is not

relationships

significantly

at 4.50 cm .s-’

where

different

could

be 1

from

the relationship

is

obviously curvilinear for both substrata. There is more variability for limpets detached at 4.50 cm .s-’ (the data points are more scattered) and linear regressions are still highly significant (r = 0.6232, P < 0.0025 for Perspex and r = 0.6396, P < 0.0004 for slate). For this reason the K values for limpets detached at 4.50 cm .s-’ were computed and used in the comparison of the different experimental separation speeds. Fig. 6 shows the variation of tenacity (K values) as a

I

-2

0

-1 LOG.

Fig. 6. Variation

function

of tenacity

of the logarithm

(K values)

SPEED

OF

with the logarithm

of the speed of pulling.

PULL,

1 cm

se;’

of the pulling

speed:

bar represents

of SF.

It shows that there is a speed at

which tenacity is maximum. For slate this speed is 0.10 cm .s-’ and for Perspex between 0.10 and 1.00 cm .s-‘. At low speed (0.03 cm .s-‘) it was observed that the foot muscles extended considerably before the limpet became detached from the substratum. Even if the hook was placed carefully on the shell, due to the position of the columellar muscle insertion on the shell, the posterior end of the foot sometimes became detached first, introducing a strong peeling component ( >60”). Even if limpets were induced to clamp their shell down against the substratum it is possible that differences in the contraction of the columellar muscle may increase variability especially at the higher speed (4.50 cm .s-‘). At intermediate speeds (0.10 and 1.00 cm .s-I), the

TENACITY

OF PA TELLA

2%

VULGA T.-l

Iimpets have time to contract fully the foot muscles and are pulled with minimal muscular extension. Earlier it was shown that the tenacity of P. vulgata is higher on rough slate than on Perspex when detached at 0.10 cm .s-’ out of water (see p. 284). The present experiment has shown that at 0.03 cm .s-‘, the tenacity on slate continues to be higher than on Perspex (t = 5.621, d.f. = 49, P < O.OOOOl),whilst at 1.00 and 4.50 cm .s-’ no significant difference between tenacities on the two substrata was observed (t = 0.429, d.f. = 21 and t = 1.420, d.f. = 42, respectively). At 2.00 cm .s~ ‘, however, the tenacity of limpets attached to Perspex is significantly higher than to slate (t = 5.351. d.f. = 36. P
Temperature variation certainly affects different physiologica processes, such as oxygen consumption, creating variations in the general metabolism of gastropods. The effect of water temperature upon the tenacity of P. ~~~~~u~~ was investigated for animals attached to Perspex and slate. Three experimental temperatures were chosen : 7, 13 and 20 “C. Limpets were allowed to acclimate at these temperatures for at least 2 wk before the tenacity was measured. The speed of pulling was 0.10 cm . SC’. The equations of regression giving the best tit curves and the K values of the linear regression forced through the origin are given in Table VIII. The comparison of K values for each temperature (Fig. 7) shows that, for limpets

TABLE VIII

Equations of regression for the best fit curve of the relationship between the force of adhesion (F) and the area of the foot (A) of Prrrellir ~,~/~f~~~~ detached in water at different temperatures: the K values of the line&r regression through the origin are also shown; I’. P. I?, as before. Water temperature Substratum Perspex

Slate

(“C)

Equation of regression

I

P

n

K i ST (kg .cm-‘)

7 13 20

F = 0.820 A - 0.008 F = 2.200 A - 1.049 F = 2.770 A + 0.617

0.5142 0.8613 0.9578

(0.024 io.001
19 39 14

0.818 I!z0.181 1.980 * 0.120 2.920 i 0.140

7

F= 2.055 A”“3’ F= 1.195 A’ ‘27 F=2.882 A - 1.93I

0.9064 0.9229 0.8250

to.001 to,001 < 0.001

12 27 23

1.930 i 0. I88 2.447+0.119 2.558 i 0.165

13 20

J.-F. GRENON

296

AND G. WALKER

on Perspex, it increased with the temperature (7-13 “C, q = 8.387; 13-2O”C, q = 5.041; 7-20 “C, q = 10.590; P < 0.001). For limpets on slate, however, variation in tenacity occurs at the lower temperatures only. A significant difference was observed between the tenacity of limpets maintained at 7 and 13 “C (q = 3.14, P -=c 0.05), but no significant difference was observed between the tenacity of animals maintained at 13 and 20 “C (q = 0.544). Furthermore, it is interesting to note that tenacity is higher on slate at 7 and I3 “C, but at 20 “C no significant difference in tenacity was observed between limpets attached to slate and Perspex (t = 1.486).

Fig. 7. Variation

of tenacity

(k’values)

with water temperature:

bar represents

rt ST.

The viscosity of liquids normally varies inversely with temperature. If limpets were adhering with the type of adhesion described by Stefan (1874) a reduction in temperature would decrease viscosity therefore reducing tenacity. It is possible, however, that because the viscosity of the pedal mucus is so high (1 x IO’ poises; see Grenon & Walker, 1980), smalt variations of temperature effect only small changes in the viscosity (or elasticity) and thus tenacity is not affected. On the contrary, metabolic processes are very sensitive to temperature variation. Davies (I 966), Bannister (1974) and Branch & Newell (1978) have shown that the metabolic (respiratory) rate of different species of limpet, like that of all other invertebrates, increases with temperature. Limpets with higher metabolic rates probably contract their foot muscles more powerfully and thus are able to increase the rigidity of the foot. Water temperature has not been considered in the more recent studies on gastropod adhesion (Miller, 1974; Warburton, 1976; Branch & Marsh, 1978) and this may explain in part some of the variation measured between species of the same genus.

TENACITY

OF PATELLA

C’GLGATA

297

Limpets, being intertidai animals, are exposed for long periods of time and so must cope with the problem of water loss (Davies, 1969). To investigate the effect of duration of emersion on tenacity, groups of Pat& vulgata attached to slate were taken out of water and kept at room temperature (20 f I “C) for 1, 2, 4, 6, 8 and 12 h before the measurement of the force of adhesion. The speed of pulling was 0.10 cm .s-‘. After 12 h no limpets had died. When the experiment was completed the limpets were replaced in tanks of running sea water and none was dead after a week. The equations of regression for the best fit curves and the K values of the linear regression forced through the origin are given in Table IX. As before, the relation-

TABLE IX

Equations of regression for the best fit curve of the relationship between the force of adhesion (F) and the surface area of the foot (A) of Parella n&m attached to slate and detached in air after different periods of emersion: r, P, n, as before. Time of emersion (ht

Equation

P

of regression _--~

I

F‘= 3.693 A - 1.158

2 4 6

F=3.129,4 F = 2 03, F = 3:542 F = 4.210 F zz 3.664

8 12

-0.14s ,,$ I.?3s A - 2.801 A - 2.399 A 0.929

0.9593 0.9764 0.943 I 0.9548 0.9689 0.9745

P _“.. _~-_-.__
< 0.001
n

K&SE (kg ~crn-‘)

~16 13 13 12 14 12

.._“.__. 3.473 3.102 3.566 3.053 3.767 3.218

_t 0.173 Lt 0.119 rt 0.199 * 0. t-59 + 0.168 zk 0.168

ship between the force of adhesion and the foot surface area is Iinear or slightly curvilinear. For those curvilinear relationships, the exponent of A is not significantly different from I (P < 0.01) and for this reason, they were reduced to linear regressions passing through the origin. No significant differences were observed between the K vaIues (SNK test) for the different times of emersion. During emersion, two main physiotogicaf changes occur: water loss {Davies, 1969) and aerial respiration. Although water loss was not mo~litored during the experiment, no significant effect on the tenacity of P. vulgara occurred over the 12-h period. Bannister (1974) showed that aerial respiration of P. lusitanica is about three times higher than aquatic respiration, but for P. caerufea aquatic respiration is twice that in air. Branch & Newell (1978) did not find any difference between aerial and aquatic respiration rates for P. ~uc~fea~. The same authors, however, found that size was an important factor governing aerial and aquatic respiration rates for P. granularis. They found that in air small P. g~ararzzdmis respire at a faster rate but

298

J.-F. GRENON

AND

G. WALKER

for larger limpets the faster rate occurred in water. The reverse was true for P. oc~dus. Baldwin (1968) found that Acmaea digitalis and A. scahra have lower rates of respiration in air than in water. Houlihan & Newton (1978) found, however, that Patella vulgata has a higher respiration rate in air than in water (at 10°C). It was shown in the previous experiments that an increase of water temperature and probably of metabolic rate increases tenacity. The fact that P. vulgata respires at a faster rate in air supposes a higher metabolic rate and it may be for this reason that K values are higher than in the first experiment (see p. 284). In the first experiment limpets were taken out of water just before the force measurement, but water was probably retained in the pallial cavity. From this experiment it can be concluded that desiccation (over the experimen~l time period) has no effect on the tenacity of P. v~~gata but the change from aquatic to aerial respiration has a marked effect. &ffkct qf’metabolic rate reduction

The previous experiments have shown that an increase in temperature, and hence an increase in the metabolic rate of P. vulgata, leads to an increase in tenacity. The following two experiments were designed to reduce experimentally the metabolic rate to see whether tenacity is affected. .Effkxs ~~a~o~ia. The effect of anoxia on the tenacity of P. ~~~~gatff was investigated by placing limpets in almost O,-free sea water, achieved by continuous bubbling of nitrogen into sea water, for different periods of time. Limpets were attached to Perspex. The water temperature was maintained at 13 “C. In this particular experiment, it was essential to use a transparent substratum in order to be able to photograph the foot of the limpets just before detachment, because it had been noticed that the area of the foot in contact with the substratum of animals kept in O,-free sea water was reduced and the formula used for non-transparent substrata was therefore not applicable. Other responses such as mantle retraction and shell elevation were also noticed during the experiment. At the start, the oxygen tension was measured as 150 mm Hg (using an oxygen electrode coupled to an acid base analyser (PHM 71 Mk 2, Radiometer, Copenhagen) and 5.5, 13,21,28 and 36 h after nitrogen bubbling the oxygen tension was down to 4 mm Hg (f 1). The experiment was not carried out for longer than 36 h because, after this period of time, mortality was high. Limpets were detached in the O,-free sea water at a pulling speed of 0.10 cm . s-’ . The equations of regression for the best fit curves and the K values of the linear regression forced through the origin are given in Table X. The equations of regression for each period of anoxia are highly significant. At 13 and 2 1 h, the best relationships between the force of adhesion and the foot surface area are curvilinear but linear regressions were significant (P < 0.001 and P < 0.02, respectively) and

TENACITY

the K values tenacity

will therefore

(K values)

OF PATELLA

VULGATA

be used for comparison.

with the time that limpets

299

Fig. 8 shows the variation

were left in anoxic

conditions.

in The

TARLE X Equations of regression for the best fit curve of the relationship between the force of adhesion the area of the foot (A) of Purellu vulgata detached after different periods in anoxic conditions: K as before. Time of anoxia (h)

Equation

0 5.5 13 21 28 36

F = F= F = F = F = F=

of regression

2.200 2.454 0.674 1.903 1.697 1.123

A - 1.049 A0.995 A ‘.5ox A0.673 A - 0.276 A + 0.823

OJ , , 0

,

, ,

r

P

n

0.8613 0.9378 0.9371 0.7534 0.8921 0.6851

< 0.001 < 0.001 < 0.001
39 20 16 14 14 12

, , TIME.

Fig. 8. Variation

of tenacity

, ,

, , ,

20

10

,

30

(fl and r, P. n,

K & SE (kg .cm -‘) 1.982 2.233 1.756 0.678 1.639 1.230

+ 0.121 & 0.134 f 0.187 i 0.108 + 0.100 + 0.168

, 40

h

(K values) with time left in anoxic

conditions:

bar represents

+ st

general tendency is for a decrease in tenacity with time. The normal response of a limpet to any stress is to clamp down its shell against the substratum (Arnold, 1959; Wolcott, 1973) by contracting the columellar muscle and this could explain the increased tenacity after 5.5 h. As time of anoxia increases, a,reduction in the metabolic rate occurs and this is likely to impair the ability of the foot muscles to contract strongly. Limpets were probably in a state of narcosis and this would explain the reduction in tenacity. It is interesting to note, however, that P. vulgatu is quite resistant to a reduction of oxygen tension, the tenacity being greater than atmospheric pressure even after 36 h .under anoxic conditions.

300

As mentioned interesting

earlier,

observation

to the substratum.

J.-F. GRENON

AND G. WALKER

if the

period

36-h

on these dead animals

Miller

(1974) reported

was

exceeded,

limpets

was that they still remained

that after immersing

specimens

died.

The

attached of Thais

lamellosa

in 02-free water (cooled, boiled sea water) they dropped off the wall of the container within an hour and concluded that energy was expended in the process of adhesion. In the light of the present experiment and the previous ones, it is possible to say that in the case of P. vulgata, energy is expended in the process of adhesion in the sense that energy is released for muscular contraction and that the limited adhesive power of dead animals must be due to the inherent tackiness of the pedal mucus alone. Effect of narcotization. Various narcotizing agents used on gastropods are known to relax muscle (Runham et al., 1965) and slow down the metabolic rate. MgClz is considered one of the most effective narcotizing agents for marine molluscs (see Runham et al., 1965) and its effect on the tenacity of P. vulgata has been investigated. Specimens of P. vulgata attached to Perspex were flooded with a solution of 10% MgClz made up in sea water and the tenacity was measured at a pulling speed of 0.10 cm .s-’ after 5 h immersion in the narcotizing solution at 13 “C. The equation of regression for the best fit curve is : F = 0.226 A ‘.“’

and r = 0.8862 (P < 0.001, n = 14). The relationship is best described as curvilinear. This shows that the force of adhesion of larger limpets is less affected than smaller ones, possibly because larger limpets take longer to become fully narcotized. A linear relationship was also highly significant (P < O.Ol), however, and for this reason the K value was computed in order to compare it with the K value of nonnarcotized limpets (see p. 287). The K value is 0.598 kg .cm-’ (+ SE 0.092) and is significantly different from the K value of non-narcotized limpets (1.982 kg .crn-‘) (t = 6.855, d.f. = 49, P < 0.001). The effect and mechanism of action of narcotizing agents on marine invertebrates are not yet fully understood. What is certain, however, is that muscles relax and as contracted foot muscles help to confer rigidity to the foot, this rigidity is reduced. Maintenance of foot rigidity, as described earlier, is essential for good adhesion. Under the influence of the narcotizing agent, not only are the pedal muscles relaxed, but also as a direct consequence the pedal blood pressure will be reduced. This part of the hydrostatic skeleton is the other main factor in maintaining foot rigidity. These two experiments do show the adhesive power of the pedal mucus alone, because the muscles and the blood system were somewhat incapacitated.

TENACITY

OF PA TELLA

YULGA 7-A

301

.!Tffkct ~~~~abitclt and verticaf distribution

In all the previous experiments of the present study, limpets were collected randomly, from exposed shores. In P. vulgata there is a phenotypic difference between animals from the lower and upper shore levels (Russell, 1907; Orton, 1928). High shore level P. vulgata grow taller shells in proportion to their length (anteroposterior); in low shore level P. vulgata the height/length ratio is smaller. The present experiment was carried out to see if the tenacity of two populations of P. vulguta (high and low shore levels) show differences and to see if there was any tenacity difference for limpets inhabiting exposed and sheltered shores. Limpets attached to Perspex were detached in air, at room temperature (20 + 1 “C) at a pulling speed of 0.10 cm . s ’ . The relationship between the force of adhesion and the foot surface area for high and low shore level P. vulgata from exposed and sheltered shores are highly significant and the equations of regression for the best fit curves are given in Table XI. The relationships are linear so the K values were computed (Table XI). The TABLE X1 Equation of regression for the best fit curve of the relationship the area of foot (A) of Putella vu!gu?u from different habitats before.

Habitat

Vertical distribution

Equation

of regression

between the force of adhesion (F? and and vertical distributions: r, P. IZ. K as

,

P

n

K ri: SE (kg ‘cm-‘)

Exposed

Low High

F= 1.368 A + 0.324 F = 1.679 A - 0.638

0.8408 0.7712


21 36

1.434 I 0.09 I 1.552 i: 0.090

Sheltered

LOW

F=

1.451 A -0.258 F = 2.065 A - 1.689

0.7795 0.8546


21 15

1.481 ~0.118 1.779 rt 0.157

High

Student t-test showed that there was no significant difference between the tenacity of low and high shore level P. ~~~~[gfftaon both exposed (t = 0.840, d.f. = 53) and sheltered shores (t = 1.509, d.f. = 32). It is then possible to compute a common K value for high and low shore limpets from sheltered habitats. The K values for exposed and sheltered habitats are 1.511 kg *cm-’ and 1.587 kg *cm-‘, respectively. Using these K values, the Student r-test shows that there is no significant difference between the tenacity of limpets living in exposed or sheltered habitats (r = 0.678, d.f. = 89). It is then possible to calculate an overall K value of 1.549 kg . cm--’ which is not signilicantly different from the value obtained in the experiment during emersion, on Perspex. The casual observations by Kitching ef al. (1966) on the adhesive power of ~uce~la iap~~~us from exposed and sheltered habitats showed that it was higher for exposed

302

J.-F. GRENON

AND G. WALKER

animals. The collected animals, however, were allowed only 5-15 min to reattach to the experimental plates. With Patelia vulgata it was noticed that tenacity was minimal after such a short period of time, Although phenotypic differences occur between populations of P. vulgata, this experiment shows that such differences do not affect tenacity. Locomotion When a limpet is detached out of water the pedal mucus is tacky when touched with a dry finger. If a wet finger is used, however> there is no tackiness. This simple but important observation holds the key to the way a limpet moves and adheres. P. vulgatn moves using retrograde ditaxic locomotory waves (Jones & Trueman, 1970). These authors also showed that a limpet when moving has only 50”5
TENACITY

functions found

in locomotion

in the centre

release of mucous

for an unspecific

time. Patches

of the trail and such mucous secretions

303

OF PA 7-L-LLA VULGA TA

from the various

Fig. 9. A MUCOUStrail (between

arrows)

of mucus

loss would

were sometimes

be made

up by the

foot glands.

left behind

by .4c~n1~tr te.ssu/rrttr

Limpets shift from the moving state to the stationary adhering state rather quickly so that the mechanism for achieving both states must show marked relatedness. Again video recordings showed that in passing from the moving state to the adhesion state, water present in the locomotory waves was quickly expelled, so that when 50’:; of the sole surface is in contact with the substratum during locomotion (see p. 302), in the adhesion

state lOOo/, is in contact.

With all the water expelled,

the highly tacky pedal mucus is brought into play in securing strong adhesion. When the animal needs to move off again from the adhesion state, a locomotory wave is formed at the anterior foot margin and the water taken up will permit the foot to be progressively released from the substratum. A further piece of evidence which favours such a system is that the pedal mucus of limpets does not flow easily (see Grenon & Walker, 1980). Easy mucous flow and low tackiness would be needed if the animals were using mucus solely for locomotion purposes.

304

J.-F. GRENON

AND G. WALKER

CONCLUSIONS

The present investigation has clearly shown that adhesion of Pateila vulgata is influenced by both physical and physiological factors. The tenacity is sensitive to surface properties of the substratum. In a natural environment, type and roughness of the substratum is variable and films of different nature (bacterial, algal, mucoid, etc.) may cause changes in the surface properties of the substratum. Different angles of detachment were tested and it was shown that when a strong peeling component was introduced, 75% of the normal force was needed to detach a limpet. Contrary to a normal pull, when a shear pull is exerted the force is not proportional to the surface area of the foot but the mean shear tenacity remains high due to shell friction on the substratum. It has also been shown that speed of separation affects tenacity; there is a speed at which tenacity is maximum. Limpets are submitted to environmental forces such as waves, currents, wind, gravity and predators. These forces will act differently on limpets, introducing different angles and speeds of detachment. It was shown that tenacity increases with rising water temperature. At higher temperatures, limpets are able to contract their foot muscles more powerfully. This indicates that an increase of foot rigidity increases tenacity. Desiccation has been shown to have no effect on tenacity but a change from aquatic to aerial respiration increases tenacity. Limpets are probably more vulnerable to predators at low tide. The increased tenacity when limpets are emersed gives them more protection against dislodgement by predators. Reduction of metabolic rate has been shown to decrease tenacity; reduction of muscle tonus is responsible for decreased tenacity. foot rigidity being essential for efficient adhesion. There was no difference in the tenacity of limpets from exposed and sheltered habitats. Vertical distribution (high and low level on shore) also had no influence on tenacity. Physical factors act on the mucous layer itself whilst physiological (metabolic) factors affect the pedal muscle system and the haemolymph pressure which maintain the foot rigid. It has been shown that adhesion as such resides in the tackiness of the pedal mucus. In the light of the results obtained in this study and those obtained previously (Grenon & Walker, 1978, 1980 and in prep.; Grenon et al., 1979) it is possible to crystallize ideas concerning the adhesion mechanism of limpets. It is concluded that adhesion can be explained fully by the tacky behaviour of the pedal mucus. As mentioned in the Introduction, tacky behaviour of various liquids has been the subject of many investigations. It is probably wiser to consider each approach to tacky behaviour separately. Can the Stefan approach explain limpet adhesion? In the Stefan approach, the tack determining factor is viscous flow of the adhesive which means that the tacky behaviour of a liquid which has an elastic component (viscoelastic liquid) cannot be explained by this approach. To see how the tenacity of limpets is far from the value predicted by Stefan, equation (12) should be used, since pedal mucus is known to be non-Newtonian (see Grenon & Walker, 1980). As mentioned by Grenon & Walker (1980): however, a yield value could not be

TENACITY

determined

for the mucus and for this reason

order of magnitude. considering

OF PATELLA

only

Considering the viscous

C’L’LGA7’,4

equation

305

(6) will be used to obtain

an

a limpet with a foot 4 cm long and 2 cm wide and element

(1.53 x IO’ poises)

the tenacity

calculated,

using 3 pm for the thickness of the mucous layer and 0.10 cm .s-’ for the speed of separation, would be in the order of 4.16 x 10” kg . cm-‘. Obviously a tenacity of this order of magnitude is never obtained with limpets. It has been stressed by Bikerman (1947) that when the viscosity becomes very high even if it continues to be a true Newtonian viscosity, Stefan’s equation loses its validity and the predicted value is much in excess of the tensile strength of the best structural materials. The Stefan type of adhesion fails to explain the adhesion of limpets. In the second approach to tacky behaviour, elasticity is considered to be the tack determining factor (Voet & Geffken, 1951). Their mechanical deformation theory seems more adequate in explaining the adhesion mechanism of limpets and indeed there are many common points. The mucus does have a viscoelastic behaviour as required by Voet & Geffken’s theory and the force of adhesion measured for limpets is much lower than predicted by Stefan. In the case of the viscoelastic theory, fracturing is preponderantly cohesive, either in the adhesive layer or in the adherend (Salomon, 1965). Cohesive failure (usually in the mucous layer) also seems to be the rule with limpets detached normal to the substratum. In purely viscous deformation (Stefan approach), the highest stresses occur during the first instant of the stress application. In elastic deformation, however, the stress increases during separation (Voet, 1965). Grenon et al. (1979) showed that in viscous deformation (glycerol) the maximum force occurs in the first instant of the stress application (0.3 s). When limpets are detached from the substratum, however, (see Fig. 6, Grenon et ul., 1979) the force increases with time, the maximum force being reached after z 5 s of the stress being applied. Limpet adhesion is therefore best explained by the viscoelastic theory, tack being due to the stored elastic energy within the pedal mucous layer. The high tackiness

of pedal

mucus

seems to conflict

with the fact that

limpets

can also move. When water is in contact with the mucus, this tackiness disappears and P. vulgara uses this property in order to move and adhere at will. A small amount of water is incorporated at the anterior margin of the foot within each locomotory

wave, and this water assists in the progressive

release of the tacky pedal

mucus from the substratum as the locomotory wave moves backwards. When a limpet wants to adhere strongly, the water within the locomotory waves is quickly expelled posteriorly so that the whole mucous layer is immediately brought back in contact with the substratum. Amongst the gastropods, limpets have the greatest tenacity but are probably one of the slowest movers. The maximum speed recorded for P. vulgata is 0.04 cm .s-’ (Jones & Trueman, 1970). This is much lower than the values given by Miller (1974) for other gastropods using the same locomotory mechanism. She gives a mean value of 0.14 cm .s-’ for those gdstropods using the retrograde ditaxic type

J.-F. GRENON

306

of locomotion. locomotory

AND

It seems that gastropods speed and vice versa.

also occurs between

limpets

Branch

G. WALKER

must

sacrifice

& Marsh

tenacity

for high

(1978) have shown

effective

that this

of the same genus.

ACKNOWLEDGEMENTS

We are grateful to Professor D. J. Crisp, for criticism of the manuscript. We also wish to thank Mr. J. Jacques for producing diverse accessories for the adhesion testing apparatus and Mr. W. Rowntree and Mr. D. C. Williams for the photographic work involved. Mr. A. Vardy (School of Plant Biology, U.C.N.W., Bangor) gave advice on statistics and Mr. A. Yule assisted with computer programming. J.-F. G. would like to acknowledge the “Societe Zoologique de Quebec” (P. Q.. Canada) for financial support during the course of this study.

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