Roy Gibbens III
Introduction
Small clonal and large solitary individuals of the
sea anemone Anthopleura elegantissima,occur predominantly
on rocks in the intertidal zone from Alaska to Southern
California (Hand, 1955). In many situations these anem-
ones cover all or part of the column with small gravel,
bits of shell, or algae. The covering materials adhere
strongly to the verrucae on the column (Hand, 1955).
The present investigation was conducted to deter-
mine how the anemones attach gravel to themselves, and
to ascertain the relation betweenithe availability
gravel and the amount of gravel actually attached to
anemones. The facilities at Hopkins Marine Station of
Stanford University in Pacific Grove, California were
used to conduct all aspects of this investigation.
Gross Morphology, Distribution,
and Number of Verrucae
The gross morphology of the verrucae of 8 clone¬
mates used in experiments to be described later was
studied. The anemones were first relaxed in Mgcl2 solu-
tion isotonic with seawater for 4 hours then individually
examined under a dissecting microscope in a bowl of sea
water.
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Figure 1 illustrates representative types of verrucae
seen, and shows how they are distributed on the anemone's
column. The large simple verrucae, characterized by
one vesicle, occurred on the proximal 3/4 of the column,
whereas the compound and the small simple verrucae were
seen only on the top 1/4 of the column. All simple ver-
rucae had at the distal tip an off white patch of tissue
distinct from the green pigmentation (located in the
ectoderm: Buchsbaum, 1968) of the surrounding tissue.
Compound verrucae bore such a patch at the tip of each
projecting vesicle of the verruca. The longitudinal
rows of verrucae, characteristic of Anthopleura elegan-
tissima, were delineated by observation and probing with
a dissecting needle. In the clone examined, all anemones
seen had numerous rows of verrucae extending from the
collar, down the column to the pedal disc. Between these
complete rows, all anemones had some rows of verrucae
which started at the collar but at some point at or above
the middle of the column terminated. These observations
are consistent with those of Hand (1955). Data on num-
bers of verrucae per longitudinal row and numbers of
complete rows of verrucae for each of the 8 clonemates
are presented in table 1. It is evident that the number
of verrucae in the longitudinal rows extending the length
of the column is not constant, either within an individual
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or between clonemates. Also, there appears to be no
correlation between the number of complete columns and the
size of anemone as measured by bouyant weight or body
dimensions. The data obtained are not complete enough
to allow further generalizations. It was not possible
to obtain quick estimates of the number of verrucae
per anemone.
Microscopic Structure of Verrucae
To study the microscopic morphology of verrucae,
frozen sections of an anemone fixed in 10% formalin sol-
ution were cut at 32 microns using an international cry-
ostat microtome. Figure 2 illustrates the salient features
of a longitudinal section of a verruca. The absence of
zooxanthellae in the gastrodermis at the very tip of
verrucae may help to explain the offwhite color of the
patches at the tips of the verrucae.
The distribution of cnidocysts around the verrucae
tip was studied. Cnidocysts were identified using il-
lustrations of the cnidom of Anthopleura elegantissima
(Hand, 1955). Nearly all cnidae observed lay in the
ectoderm; exceptions were 7 spirocysts observed in
zooxanthella laden gastrodermis when the tissue was first
resolved, and 9 spirocysts that were resolved in the
gastrodermis at the tip of the verrucae in an area that
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appeared to be a pore. Under oil immersion counts were
taken of cnidocysts in the ectoderm in the field of view at
the tip, middle and base of the verrucae. Another ver-
ruca tip in the same section was also counted (Table 2).
Only spirocysts and basitrichs were identified in this
section. The counts indicate there is a gradient in the
density of spirocysts in the verrucalar ectoderm. The
density is greatest in a ring at the verruca tip. Hand
(1955) indicates there are cinclides at the tips of the
verrucae in Anthopleura elegantissima which could account
for the indication of a pore seen in one section (not
illustrated).
(195
Hand'describes the cnidom of the column as consisting
of atrichs and basitrichs. He does not mention the
presence of spirocysts on the column, but he did not
examine the cnidom of verrucae specifically. In addition
to spirocysts some basitrichs were identified in the
sections of verrucae. No other types of cnidocysts were
observed.
Mechanism of Attachment
The fact that occassionally verrucae tips were
torn off when gravel cover was plucked from an anemone
indicates how strongly verrucae can adhere. Some obser-
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vations and simple experiments were conducted to throw
light on the mechanism of adhesion.
Verrucae were probed at various points with a dis-
section needle and the only tissue noted to be readily
adherent was allthe whitish patchs at the tips of ver-
rucae. Next, numerous small fragments of cover slips
were presented at verruca tips, allowed to attach, and
then pulled off and examined microscopically. The best
fired
of such preparations revealed a mass of cnidocysts. By
cutting off a verucca tip attached to a cover slip a
preparation was obtained which showed both the attach-
ment spot on the cover slip and the verruca tip itself,
separated by a cleft more than 500microns wide which was
spanned by a mass of cnidocyst threads (figure 3).
The only possible explanation for the physical attach-
ment of the tissue to the cover slip was the mass of threads
extending from tissue to glass. Because true nemato-
cysts were scarce here, and the mass consisted very largely
of partially fired spirocysts and empty spirocyst cap¬
sules, there can be no doubt that it is the spirocysts
which enable verrucae to adhere to objects.
Other cases of cnidocyst adhesion have been docu-
mented. Mariscal (1972) found that basitrichs were the
primary nematocysts enabling the sea anemone Calliactus
tricolor to attach its tentacles to shells of hermit
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crabs on which it settled. Mariscal also observed spiro-
cysts and prepared a scanning electron photomicrograph
of the thread of a discharged spirocyst. He noted that
a web of microfibrillae extends from the shaft of the
discharged spirocyst thread and noted that this feature
might enable spirocysts to function in an adhesive cap-
acity. Anthopleura elegantissima has spirocysts in
other parts of the body, but spirocysts attach strongly
to foreign objects only at or near the verrucae tips.
(Occassionally gravel was noted to adhere to tentacles,
but not for more than a fraction of a second) Spirocyst
depletion and the occurence of a cinclide in verrucae
tips might help explain the sharp decline in spirocyst
number of the very tip of the verrucae shown in Table 2,
Two other observations were made incidental to the
above studies. First, gravel presented to an anemone
denuded of cover adhered better after 24 hours than it
did only an hour after it attached. Perhaps more time
allows more spirocysts to attach. The observation sug-
gests that the longer gravel is attached the better it
adheres. However this may not be the case. A second
observation suggests the attached life of a piece of
gravel may be limited. Seven anemones collected in the
field and transferred to plastic tanks with slowly
running seawater and constant illumination lost much of
Roy Gibbens III
their adhering gravel the first week in lab, but still
retained some after 3 weeks. No studies of turnover
rate of covering material were carried out.
Relation between Gravel
Availability and Amount of Cover
Preliminary observations
In the field, Anthopleura elegantissima were seen
bearing some gravel cover whether they were on horizontal
or vertical rock faces. In the laboratory it was obser-
ved that anemones collect small gravel on the column if
they are simply buried in it or if it is presented to
them in a fast moving current of water. The latter
observation suggests that wave action can make water
borne gravel and shells available to anemones regardless
of their orientation. Wave borne gravel appears to be
the only source of many anemones in the intertidal zone.
It was interesting to observe that some anemones sub-
jected to the scouring action of gravel in a water current
in the lab swelled their verrucae shortly after exposure
was begun. The same response was observed in anemones
that contracted when they were probed several times with
a dissecting needle. The swelling of verrucae in response
pattern
to abrasion would seem to be'behavior'facilitating gravel
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pick up. Other than this behavior, throughout the course
of this study, Anthopleura elegantissima was never ob-
served to actively "reach" for gravel with
tentacles
or by bending the column down to contact the substratum.
Field observations suggest that gravel cover present
on anemones is related in part, at least, to the avail-
ability of gravel. Anemones observed at the water level
on the Monterey wharf pilings several meters above the
bottom are essentially bare of gravel cover. Only an
occassional anemone here
had a few bits of attached
shell. At the other extreme,anemones on flat rocks
lying low in the sand and subjected to much washing by
surf, are profusely covered by sand gravel. In many
places along the Monterey peninsula it was seen that
anemones on rocks more distant from beds of sand had
observably less gravel cover than anemones that were
closer to a source of gravel. It seems reasonable that
a difference in gravel availability might account, at
least in part, for the difference in amount of cover
observed on anemones. To test this hypothesis 3 exper-
iments testing different aspects of availability were
conducted.
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Experimental Methods
Forty-three anemones all clone-mates and denuded of
sand, were established on separate plastic petri dish
lids 5 cm in diameter. To facilitate adhesion of the
anemones, fine sand was glued to the surface of the petri
dish lids with 5 minute epoxy and allowed to dry. A
loop of nylon thread was tied to a hole in the edge of
each lid so that anemone and dish together could be easily
weighed. an anemone was placed on each lid, allowed to
attach, and was dept on the lid through all subsequent
operations. The lids with anemones fitted loosely in
the bottom of 150 ml beakers measuring 5.5 cm X 8 cm.
Animals were thus maintained in separate beakers provided
with running seawater between experiments.
Sand weighed to the nearest 0.1 g bouyant weight
using a beam balance was poured in along the side of
the beakers without touching the anemones. It was made
available to the anemone by agitating the water and gravel
in the beaker with clockwise and counter-clockwase motions
of a jet of sea water directed through a retainer screen
at the top of each beaker. The water pressure was stand-
ardized by adjusting the water flow so that from a heighth
of 3.5 cm the jet from the 3/8 inch nozzle used shot
16 cm up the 3 degree incline of the sea table on which
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the experiment was performed. The standard sand used
in the experiment was sifted by a 2.00mm mesh screen
and retained by a.991mm mesh screen. Eleven samples of
25 sand grains each were counted out and their bouyant
weights taken. Weights for the 25 grain samples ranged
from 107-140mg: mean 119mg, standard deviation 8.25,
standard error of the mean 2.49. It can be judged that
the error due to gravel bouyant weight variance intro-
duced in measuring gravel cover by bouyant weight was
not appreciable. Because the water content in anemones
varies with their degree of contraction, all weights
reported are bouyant weights determined to the nearest mg
by immersing the anemone plus petri dish lid to which it
adhered in a beaker of sea water. The plate was suspended
by its nylon thread from a torsion balance. Initial
weights of the anemones were taken before the experiment.
The ability of each anemone to cover itself maximally
with gravel was determined at the end of all experiments
in which that anemone was used. This was done by burying
the anemone in gravel and comparing the weights of the
anemone without cover and with maximum cover. Counts
of the number of exposed bare verrucae were taken on
each anemone when it had attained maximum gravel cover
in order to express what further capacity the anemones
had to attach gravel. The greatest count of bare verrucae
10
Roy Gibbens III
for any such anemone was 8, indicating animals attained
virtually 100 % cover. All experimental results are there¬
fore reported in terms of percent of the maximum gravel
cover attained (bouyant weight).
The relation between anemone bouyant weight and
bouyant weight of maximum possible gravel cover for every
anemone used in experiments is shown in Fig. 4. The
data support a general positive correlation between
the size of an anemone and the amount of gravel it can
carry.
rst experiment
This experiment was conducted with 14 anemone to
test whether or not, other things being constant, there
is a relation between frequency of exposure and amount
of cover an anemone bears. In the experiment anemones
were exposed intermittantly to agitated water and sand.
1.5g bouyant weight of gravel was added to each beaker
and agitated as described for one minute. Gravel re-
maining loose after one minute was poured out of the
beaker and the anemone with its gravel cover was weighed.
The exposure procedure was repeated at one hour inter¬
vals 5 times. In between exposures the animals were
kept in beakers of gently flowing seawater.
Roy Gibbens III
During the experiments it was noted that some anem-
ones were attached at the centers of their plastic lids,
others near one side. Because anemones near the sides
of their lids were observed to be subjected to more scour
of gravel than anemones in the centers of their lids,
results are reported according to the position of the
anemones on the lid in Fig. 5.
Figure 5 shows the % of maximum gravel cover attained
by anemones on the centers and sides of lids after each
exposure. A composite curve for all 14 anemones, with
means, ranges, and standard deviations is shown at the top,
The T value significance levels of the differences in the
percent of maximum gravel cover on anemones classified
as center and side is presented in Table 3. T value
significance levels for the differences in cover between
exposures for side, center, and composite groups are
also tabulated.
The difference in Zmaximum cover attained by side
and center anemones is statistically significant fol-
lowing the first 2 exposures, but does not satisfy the
95% criterium for significance after the last 3 exposures.
In general, the composite graph for the 14 anemones
represents the gravel cover gained by this clone of an-
emones after each exposure. The percent of maximum gravel
12
Roy Gibbens III
attained increases with the first two exposures but later
levels off at a value less than half the maximum gravel
cover capacity of these anemones. Considering the fluc-
tuations in gravel cover in individual anemones it is
evident that anemones lost gravel as well as gained it
in successive exposures. As noted previously, verrucae
seem to adhere more strongly to gravel given more time
for attachment. It seems likely therefore, that the
surge of water the anemones were subjected to could act
to detach loose gravel recently picked up.
Second experiment
This experiment was conducted to ascertain if,
other things being constant, there was a relation between
the duration of exposure to gravel and the final amount
of gravel cover. For this experiment 7 anemones were
exposed only once to agitated seawater and gravel for
intervals of 0.5, 1, 2, or 4 minutes. Two grams of gravel
(bouyant weight) were used in each exposure.
The results are shown in Fig. 6. T value signi-
ficance levels (Table 4) indicate that the increase in
gravel cover shown between animals exposed for 0.5 minutes
and those exposed for 1 minute is statistically signi-
ficant. After 1 minute of exposure there is no signi¬
ficant change in the amount of covér. The results of
Roy Gibbens III
this experiment are consistent with the results of Ex-
periment one: gravel cover increases with greater avail-
ability until an upper limit seems to be reached, but
this upper limit is scarcely a third of the maximum
gravel-carrying capacity of the animals tested.
Third experiment
In the third experiment gravel availability was
varied by varying the amount of gravel presented to
anemones during exposure of fixed duration. Using 0.5.
1, and 2 grams (bouyant weight) of gravel, three groups
of 6 to 8 anemones were exposed to agitated gravel and
seawater once for 4 minutes. The results are shown in
figure 7. The T value significance levels (Table 5)
indicate the change in percent maximum gravel cover at-
tained between anemones exposed to 0.5 and 2g of gravel
is statistically significant. This result quantitatively
supports a positive correlation between the amount of
gravel in the water and the amount of gravel an anemone
covers itself with under the conditions of the experiment.
14
Roy Gibbens III
Discussion
The indication from these experiments is that the
ability of an anemone to capture gravel bits with its
verrucae is directly related to the availability of
gravel to the anemone. Considering that spirocysts are
required for adhesion of gravel to verrucae, the opportun-
istic nature of this correlation can be understood.
None of the experimental animals came close to achieving
100% gravel cover under the experimental conditions used,
The experimental results do indicate that the method
of presentation may affect the amount of gravel an an-
emone attaches to itself. It seems anemones that have
a longer time to attach to gravel before being subjected
to conditions which act to remove loosely adhered gravel
should cover themselves more completely.
It is conceivable that in the field after a wave
showers anemones with sand, much gravel will settle on
and around the anemones. This mode of presentation of
gravel would seem to allow more time for adhesion than
the few moments an anemone has if sand is supplied only
during short surges of the surf, which my experiments
were designed to simulate.
Another aspect of gravel cover that still needs to
be investigated is turnover of attached gravel. The
15
Roy Gibbens 111
experimental data and observations indicate that anemones
do loose attached gravel, and so to effectively evaluate
the relation between gravel availability and amount of
cover, this parameter must be taken into consideration.
Summary
1. Gravel and other debris is often attached to the tips
of verrucae on the sea anemone, Anthopleura elegantis
sima.
2. Small simple and compound verrucae occur on the distal
1/4of the column; only large simple verucae occur on
the proximal 3/4 of the column. Verrucae occur in longi¬
tudinal rows; some rows extending from the top of the
column to the base, other rows are present only near
the top. The numbers of verrucae in complete longitudinal
rows varies around the column of an anemone.
3. Gravel adheres only to the tips of verrucae. Many spi-
rocysts and some basitrichs occur in the ectoderm of
verrucae; the density of spirocysts is greatest near
the tips of verrucae, declines at the verrucal sides,
and approaches zero near the base. Spirocysts enable
verrucäe to adhere to objects, perhaps by means of
microfibrallae extending from the shafts of everted
threads.
16
Roy Gibbens 111
4. Only gravel contacting verrucae directly is available
for capture. Gravel can be picked up in a fast moving
current of water. Wave borne gravel appears to be the
only source of cover to many anemones in the intertidal
zone. Anemones poked or subjected to scouring by water
borne gravel swell their verrucae. Anemones were never
observed to pick up gravel with their tentacles of by
bending the column down to contact the substratum.
5. In many places anemones on rocks distant from beds of
sand had less gravel cover than anemones closer to a
source of gravel.
6. Experiments with anemones exposed to gravel carried
by rapidly swirling water (simulating waves and cur-
rents) show that: (1) gravel cover increases with
successive exposures to scouring with gravel, (2) with
longer duration of exposure to gravel, and (3) with
greater amounts of gravel present in the scouring
water. Presenting gravel to anemones in rapidly mov-
ing water seems to limit the degree of gravel cover
they can acquire. The mode of presentation of gravel
seems to be related to the amount of gravel an anemone
attaches to itself.
17
Roy Gibbens 111
Acknowledgement
I would like very much to thank Dr. Donald P. Abott for
his generous help and patience throughout this investiga-
tion.
18
Roy Gibbens 111
Figure Captions
1. Distribution of simple and compound verrucae on the
column of Anthopleura elegantissima.
2. Longitudinal section of a verruca. Cnidocyst counts
were taken at sites A- (table 2).
3. Verruca tip adhering to glass.
4. Relation between maximum gravel cover and anemone
bouyant weight.
5. Relation between frequency of exposure and amount of
gravel cover acquired.
6. Relation between duration of exposure and final gravel
cover.
7. Relation between the amount of suspended gravel pre-
over
sented and the final amount of gravel'acquired.
Roy Gibbens III
Table Captions
1. Distribution of verrucae in 8 clonemates of Anthopleura
elegantissima.
2. Number of cnidocysts per field of view.
3. Statistical significance of the difference in percent
of maximum cover between groups and between successive
exposures to gravel.
4. Statistical significance of the difference in percent
of maximum cover between exposures of varying duration.
5. Statistical significance of the difference in percent
of maximum gravel cover acquired during 4 minute expo¬
sures to different amounts of suspended gravel.
20
Roy Gibbens III
References Cited
1. Buchsbaum, V.M. (1968). Behavioral and physiological
responses to light by the sea anemone Anthopleura
elegantissima as related to its algal endosymbionts.
Original manuscript. 27.
2. Hand, C. (1955). The sea anemones of central California
part II. The endomyarian and mesomyarian anemones.
Wasmann J. Biol. 13. 54-61.
3. Mariscal, R.N. (1972a). The nature of the adhesion to
shells of the symbiotic sea anemone Calliactus tricolor
Leseur. J. Exp. Mar. Biol. Ecol. 8, 217-224.
21
Anemone
Bouyant
Weight
(mg)
28
29
29
32
32
55
Oral Disc
Measurements
(mm)
Minor
Major
Axis
Axis
12
10
15
12
16
10
13
18
18
12
21
17
Table
Column
Height
(mm)
10
of Verrucae
Per Row
com. incom.
16.14 --
18.1
17
16.13
16.15 -
15.14 9.7
11
17.15
12
——
17.17
16.16
18,17 -
+ of Com.
ROwS
34
35
32
32
42
37
Position on
Verruca
Table 2
Verruca Tip A
Verruca Tip B
(figure
Spirocysts Basitrichs Spirocysts Basitrichs
--
—
—
11
30
52
10
30
58
—
12
——
Table 3
Side vs. Center
2 3 4 5
Exposure
T Value
99 99 75 83 68
Significance (2)
Level
Composite
Exposures
1-2 2-3 3-4 4-5 1-5 2-4 2-5
T Value
92 44 45 51 100 82 87
Significance (2)
Level
Center
Exposures
1-2 2-3 3-4 4-5 1-5
T Value
Significance (8)
80 91 21 34 100
Level
Side
Exposures
1-2 2-3 3-4 4-5 1-5
T Value
Significance (2) 94 5 42 12 96
Level
Table 4
Time of Exposures (min.) o.5-1 1-2 2-4 0.5-4
T Value Significance
99 7 64 100
Level (%)
C
0
lravel Bouyant
Weight (g)
Value
Significance (8)
Level
Table 5
O.5-1 1-2 0.5-2
95 9.
Figure 1
4
599
616


5 O .
800060
000 0 8
000
900
O° 06
900
60
°00
oo
o 00
0
1.0 mm
Compound
Verrucae
AN
Simple
Verrucae
A
O.6 mm
Figure 2
pidermis



zooxantheilae
— Mesoglea



Gastrodermis
Glass
Thread —
Capsule —
Flgure 3
500 mierons


Tissue











Partially
Fired
Spirocyst
14 mierons
Bouyant Wt. of
Max. Gravel Cover
o0
500
Figure 4
...
.** ..
..
. *
. ..
Anemone Bouyant Wt. mg
80
% Max. Gravel Cover
Composite
Range

Center

Side
40

Sta.
Dev.


Figure 5
Mean



Exposure Trial

Mean
% Max. Gravel Cover
0.5
20
Minutes of Exposure
40
Figure 6
% Max. Gravel Cover
100
Figure 7


0.5
2.0
Gravel Bouyant Wt.g