DeMont: Attachment in Anthopleura elegantissima
INTRODUCTTON
Ellis, Ross and Sutton (1969) demonstrated evidence for an adhesive
mucus substance on the pedal disc of the swimming anemone Stomphia coccinea.
Settling is apparently associated with the expulsion and discharge of adhe-
sive nematocysts of the pedal disc (microbasic p-mastigophores).
No previous study has been published on the mode of attachment of
Anthopleura elegantissima. I herein report on experimental findings pertaining
to mechanisms of its attachment.
GENERAL LABORATORY OBSERVATIONS
The muscular apparatus for locomotion, as described by Pantin (1952)
for Metridium, is essentially the same as that responsible for peristaltic
waves in the column. According to Pantin, a wave of contraction of circu¬
lar ectodermal muscle-fibres runs assymetrically inward from the rear edge
of the foot, causing release of the substratum. At the same time, down-
ward peristalsis in the column forces water into the foot causing expansion
there. The front edge of the foot thus advances after the pedal wave has
traveled across it.
In this investigation, an animal was placed on its side in a water-
filled glass dish. Verrucal and minor pedal disc attachment were observed.
The tentacles displayed sweeping movements across the substrate, and some
apparently attached to the glass. Different lobe-like portions of the
pedal disc became affixed to the glass as the anemone assumed an erect
posture.
Detachment was observed with the aid of the dissecting microscope
adjusted such that the animal was seen from below. Tapping the substratum
with a blunt instrument induced the pedal disc to peel off gradually
DeMont: Attachment in Anthopleura elegantissima
from the outer edges inward. The pedal disc was found to be composed of
tiny finite portions of tissue which stretched and snapped free as detach-
ment proceeded.
METHODS and RESULTS
Abilities to Attach to Various Substrates
Anthopleura elegantissima's capacity to attach to the following sub-
strates was tested: 1) Marine rock, 2) Cemented gravel and shell fragments
(cemented in paraffin wax), 3) Loose gravel and shell fragments, and 4) Porous
screen (3 meshes/mm). Anemonies of the same clone were used to prevent
genetic variations and possible aggresion. The anemonies were placed upside-
down on each substrate in aquaria supplied with running sea water. Recorded
was the time elapsed until the anemone was attached in an upright position.
The eight Anthopleura elegantissima on marine rock settled most rapidly,
with a range of seven to thirty-five minutes in attachment time. The four
anemonies on the cemented gravel-shell substrate ranked second in settling
ability, the range being one and one-half to four hours. Settlement on
the screen also occured, although the attachment time was longer, extending
from forty-five minutes to ten hours for eight anemonies tested. None of
the four anemonies on the loose gravel-shell substrate were able to attach.
Substrate Selection
Submerged anemonies were placed along the line of contact of two
rectangular plates of differing materials. The substrate selected by an
anemone was taken as the material on which it was found after twelve hours.
First considered was the choice of marine versus non-marine substrata.
Two asbestos plates were used, one soaked for several months in the inter-
tidal waters, and the second unsoaked. The intertidal asbestos appeared
roughened by the build-up of organic materials. The unsoaked asbestos, by
DeMont: Attachment in Anthopleura elegantissima
comparison, was smooth. Of eight animals tested, seven chose the marine
asbestos and one showed no movement toward either substrate; this result
was significantly different from random (p(.05; chi-squared test).
Glass plates of three textures were arranged in pairs as in the
previous experiment. The substrates tested were the three combinations of
smooth, semi-rough and rough glass adjacent to each other. Two adjoining
smooth plates served as a control (Table 1). The textural combinations of
glass substrata indicated no preference for a roughened substrate over a
smooth.
Modes of Attachment
Four factors possibly aid in pedal attachment of Anthopleura elegan-
tissima: 1) Nematocysts, 2) Substrate conformation, 3) Suction, and 4) Ad-
hesion.
Nematocysts
Frozen longitudinal sections 48 microns thick were cut from the pedal
disc of A. elegantissima with the International Cryostat, Model CTI. The
sections were then placed on slides and stained with neutral red for
histological observation. Fewer than twenty nematocysts were observed in
each of four complete sections. The nematocysts, all undischarged, were
found in the ectodermal, mesogloeal and gastrodermal tissues.
Forceful removal of an anemone from a glass slide left behind damaged
tissue which was investigated microscopically. An average of eight nema-
tocysts were found in each of sixteen random fields 144 microns in diameter.
with less than.7 nematocysts discharged per field. In contrast, an
anemone removed by tapping a glass slide left no remnant tissue behind.
Only two nematocysts were found in sixteen random fields of this preparation.
Individual anemonies were subjected to turbulent and still water condi-
DeMont: Attachment in Anthopleura elegantissima
tions to compare modes and rates of attachment. The apparatus simulating
turbulence consisted of a siphon delivering two liters of sea water per
minute into the base of a round plastic 500 ml container. The container,
open at the top, was in a constant state of overflow and caused the experi¬
mental animal to be continually tossed and turned. The still water environ-
ment was established by converting a similar plastic container into a
revolving drum. Oriented sideways and connected to a small motor, the con-
tainer revolved smoothly at 1.2 rpm. Water and anemone entered and left
through a corked hole at one end of the otherwise sealed drum. Attachment
time was recorded, time O being that time the anemone, dropped into the
water of each container. Attachment time was defined as the time at which
continuous attachment was observed for a minimum of ten seconds. In the
revolving drum, an anemone attached when it stuck to the side wall, revolving
with it.
In the turbulent environment, the anemone's mode of attachment was
difficult to ascertain due to the rapid tossing and tumbling action of the
animal. Attachment was accomplished entirely by the tentacles or by the
side of the anemone (Tentacles: 40%; Side: 60%; n - 36). Side attach-
ment may have involved tentacles, verrucae, pedal disc, or a combination
of the three. The pedal disc never served as the sole site of attachment
in the turbulent water environment. In the still water conditions of the
revolving drum, each animal fell to the bottom oriented on its side with
the plastic wall of the container moving slowly underneath. No tossing or
turning of anemone was observed. In a side orientation, the surface area
of verrucae in contact with the substrate far exceeds that of the tentacles
or pedal disc. The attachment rate of anemone is dramatically faster in
the still water environment than in the turbulent, as demonstrated in
Figure 1.
DeMont: Attachment in Anthopleura elegantissima
Substrate Conformation
Pedal disc tissue of A. elegantissima both molded to and wrapped
around individual fibers of the 3 mm mesh screens. Random movement of
suspended particles inside the molded lobes of pedal disc was observed
with the aid of a microscope.
Submersion of an attached anemone in calcium-free sea water for
several hours relaxes all musculature (Ross, 1960 and Williams, 1975). Des-
pite such relaxation, the anemone maintains firm attachment.
Suction
In a suction cup mechanism on smooth substrate, contraction at the
rim conceivably draws the center of the cup up from the substrate, reducing
the inner pressure relative to that of the surroundings,
Anemonies attached firmly to glass plates were subjected to reduced
atmospheric pressures (-707 mm mercury) in a vacuum chamber. A submerged
anemone with a pedal surface area of 6.45 square em at a pressure of -707 mm
would theoretically require approximately 500 grams of weight to be hung
normal to it before detachment would occur. Such a calculation assumes
that perfect suction is involved in attachment.
One hundred grams of weight was hung normal to an inverted anemone of
pedal surface area 6.5 cm in such a vacuum. The weight hung from a thread
passing through the lower column. At the reduced pressure of -706 mm, firm
attachment was observed.
Adhesion
Indirect observations implying adhesion as a mode of attachment are
presented in earlier sections of the paper. The cohesive intermolecular
forces of mucus to mucus and adhesive forces of mucus to substrate are
sub-processes in such a mechanism of attachment (Nachtigall, 1974). Direct
comparison of the cohesive strength of pedal mucus and sea water was under-
DeMont: Attachment in Anthopleura elegantissima
taken with the aid of the apparatus of Figure 2. A film of the mucus was
spread between two perpendicular microscope slides by rubbing the pedal
disc across the base slide. Sea water was used as a comparison. Film
thickness of sea water and mucus was controlled by placing a constant
weight of 500 grams on the top slide for two minutes prior to measurement.
line connected the top slide to the plastic weight bag at the oppocite end
of a pulley system. The tensile strength of each film (surface area 6.45 cm*)
was recorded as the weight required to pull the top slide off the secured
base slide. Gram weight, in the form of water in the plastic bag (buret-
controlled), was then converted to units of tensile strength (force/unit area).
Recordings of tensile strength present means of 9025.88 dynes/cm for sea
water, and 10,994.23 dynes/cm for pedal mucus. This difference in the
means is significant (Student's t-test: p (.02).
DISCUSSION
A survey of local intertidal Anthopleura elegantissima revealed none
attached to loose gravel and shell fragments. Observations on abilities
of anemonies to attach to various substrates likewise demonstrated A. ele-
gantissima's inability to attach to a loose gravel-shell substrate. Success-
ful attachment to porous screen indicates that large scale suction involving
the pedal disc acting as a single raised cup does not occur. On screen,
the rim of the pedal disc is unable to maintain a pressure differential
due to the substrate's porousity. If suction is a mechanism in attachment,
the cups must be numerous and small, each in contact with an individual
strand of screen material.
Tests on selectivity of marine versus non-marine substrates suggest
that the chemical composition of the substrate is involved in preferential
attachment. The roughened texture of the marine over the non-marine
DeMont: Attachment in Anthopleura elegantissima
material may also indicate the anemone's preference for an irregular
substrate providing a large surface area for maximum pedal substrate con-
formation. Microscopic observations of the base of anemonies settled to
screen support the theory of substrate conformation as an attachment
mechanism.
Studies of substrate preference utilizing degrees of roughened glass
appear to de-emphasize the importance of substrate conformation. The tex-
tural combinations of glass substrata indicated no preference for a roughened
substrate over a smooth. Glass being an artificial substrate for an anemone,
selection behavior cannot be considered analogous to behavior on natural
substrates.
The abundance of nematocysts in the anemone's tentacles and verrucae
relative to the few observed in sections and scrapes suggests that the
role of nematocysts is minor in the pedal disc. Subjected to turbulent
water, the tentacles have the capacity to initiate the attachment process,
The pedal disc, however, did not initiate attachment in the thirty-six
observed trials. While these studies cannot negate the involvement of
nematocysts in pedal attachment, it appears that their role in attachment
can only be minor. The pausity of nematocysts in the pedal disc tends to
support the concept of their minor importance.
Suction, as an attachment mechanism in A. elegantissima, is an un-
answered question. If musculature is involved in creating suction, and if
a calcium-free solution relaxes all musculature, the anemone would be
expected to release if suction is solely involved. Substrate conformation
cannot be operating on a smooth glass plate. The firm attachment does not
exclude suction as a mechanism, but suggests that suction alone cannot be
operating in attachment.
Studies on the cohesiveness of pedal disc mucus and sea water demonstrate
DeMont: Attachment in Anthopleura elegantissima
that pedal mucus is significantly more cohesive a substance than sea water.
Considering the subprocesses of cohesion and adhesion in attachment, a more
cohesive material can create a firmer attachment, adhesive and substrate
effects held constant. As different thicknesses of film create different
strengths of cohesion, optimal spacing of the pedal disc and its mucus rela-
tive to the substrate may be operating in attachment. Such substrate con-
formation in adhesion may therefore play a dominant role in attachment,
SUMMARY
1) Four factors possibly aid in pedal attachment of Anthopleura elegantis-
sima: 1) Nematocysts, 2) Substrate Conformation, 3) Suction, and 4) Ad-
hesion.
2) The pedal disc mucus is significantly more cohesive a substance than
sea water.
3) Optimal spacing of the pedal disc and mucus in substrate conformation and
adhesion may play a dominant role in attachment.
4) Large scale suction involving the pedal disc acting as a single raised
cup does not occur.
5) The pausity of nematocysts in the pedal disc suggests that their role in
attachment can only be minor.
The chemical composition of the substrate may be involved in preferen¬
6)
tial attachment.
ACKNOWLEDCMENTS
This work was done at Hopkins Marine Station of Stanford University,
I wish to thank Dr. Robin D. Burnett and Nathan R. Howe for their invaluable
advice and assistance throughout the period of research. Thanks are also ex-
tended to Dr. Donald P. Abbott, Dr. John H. Phillips and Charles H. Baxter for
DeMont: Attachment in Anthopleura elegantissima
their advice, and to Richard Haff and John Kono for their generous tech-
nical assistance.
REFERENCES
Ellis, V. L., Ross, D. M. and Sutton, L. 1968. The pedal disc of the
swimming sea anemone Stomphia coccinea during detachment, swimming,
and resettlement. Can. J. zool. 47(3): 333-342.
Nachtigall, W. 1974. Biological Mechanisms of Attachment. New York.
Pantin, C. F. A. 1952. The elementary nervous system. Proc. Roy, Soc..
Ser. B 140, 147-168.
Ross, D. M. 1960. Effects of ions and drugs on neuromuscular preparations
of sea anemonies. J. Exptl. Biol. 37: 732-774.
Williams, D. 1975. Evidence for calcium as the spike ion in the nervous
system of Metridium senile. Unpublished.
DeMont: Attachment in Anthopleura elegantissima
FIGURE CAPTTONS
Figure 1
Rate of attachment in turbulent and still water (continuous attachment
for minimum of 10 seconds) A. Turbulent water; n=62. B. Still water;
n-36.
Figure 2
A. Pulley apparatus measuring tensile strength of pedal mucus and sea
water. A) Stage supporting perpendicular microscope slides (pedal and
sea water films between slides), B) Pulley, C) Water-filled buret
adding weight to D) Plastic weight bag.
B. Enlarged overview of supporting stage and perpendicular slides. Base
slide is immobile, with two ledges preventing twisting of top slide.
Top slide in contact with base slide and line.
11
DeMont: Attachment in Anthopleura elegantissima
TABLE CAPTIONS
Table 1
Substrate selection with combinations of two adjacent glass plates of
smooth, semi-rough and rough textures. Numbers indicate counts of
anemonies found on each substrate after 12 hours, N = 18 in each
combination. The control is two adjacent smooth plates.
EXPERIMENTAL
COMBINATIONS
Control
DeMont: Attachment in Anthopleura elegantissima
TABLE 1
SUBSTRATE
Smooth
Semi-roug
Rough
10
7
7 : 10
No response
DeMont: Attachment in Anthopleura elegantissima
FIGURE 1
60
30 60 90 120 150 180 210 240 270 300
ATTACHMENT TIME (10- SECOND INTERVALS)

120
150 180 210 240
270 300
5
ATTACHMENT
TIME (10- SECOND INTERVALS)
DeMont: Attachment in Anthopleura elegantissima
FIGURE 2
E


A