ABSTRACT
The high intertidal limpet, Collisella digitalis,
was kept continuously washed by sea water and compared
with adjacent individuals under ambient conditions to
determine whether prolonged submersion constituted a
stress. The osmolarity of extracorporeal water and
body fluid were monitored over four days, along with
the force required to remove the limpets from the rock.
The tenacity dropped markedly after nine hours of
submersion and by four days was less than 503 of
control animals. Increased tenacity was correlated
with elevated osmolarity of body fluid. It is
suggested that C. digitalis behaviorally regulates its
body fluid at elevated levels and that prolonged
submersion may constitute a physiological stress and
set a lower boundary for adults of the species.
Introduction
The limpet Collisella digitalis inhabits vertical
rock faces and crevices high in the intertidal zone.
Although C. digitalis are frequently splashed during
high tide, they are rarely submerged and have the
highest intertidal distribution of central California
limpets. Breen (1972) showed that C. digitalis
exhibited upward migratory movement during the fall and
winter months coinciding with periods of heavy surf.
Observations in aquaria show that C. digitalis quickly
moves above the water line. The fact that C. digitalis
flee from sea water for respiratory reasons seems
unlikely because of studies done by Baldwin (1968)
which show high oxygen consumption during periods of
submergence.
Inspection of intertidal sites reveals many
potential sites lower in the intertidal area where they
would not seem to be excluded by competition. To
determine if C. digitalis is osmotically stressed by
sustained exposure to sea water is the subject of this
The index of stress used is the limpet's
paper.
tenacity on the substrate and this is correlated with
osmolarity of extracorporeal fluid and body fluid.
Materials and Methods
To examine the effects of submergence on tenacity.
extracorporeal fluid and body fluid osmolarity, a popu-
lation of C. digitalis on a rock face were enclosed in
a 1.2x1.2-meter fence of 1/4-inch hardware mesh. This
served to keep the limpets from escaping a constant
flow of sea water piped to the top of the study area
during the experimental period. An adjacent group of
limpets at the same tidal height were used as controls
and exposed to the normal tide and wave cycle. Before
the experimental limpets were submerged, 10 control
limpets were sampled and 10 of each control and experi¬
mental limpets were sampled 3, 6, 9, 25, 49, 73 and 97
hours after submergence.
To measure tenacity, wire loops were fixed on the
limpets' shell with splash zone epoxy compound. A
spring scale was attached to the loop and the limpets
were pulled at a 45-degree angle to approximate the
vector sum of lift and drag forces. The force (lbs.)
required to remove the limpets from the rock was
converted to Newtons and divided by the area of the
limpets' foot to obtain a value of tenacity (N/m2).
Area of the foot was calculated from the length, using
the regression equation (Hahn and Denny)
2.24
y - 3200 x
where x is the length of the limpet in centimeters and
y is the area of the foot in square meters.
Extracorporeal fluid was obtained from each limpet
by gently pressing the foot and pipeting the expressed
fluid. The osmolarity (mOsm) was assayed with a Preci-
sion Systems microsmometer utilizing freezing point
depression. This required a 50-microliter sample size.
Body fluid was collected from each sample group by an
incision through the sole of the foot and extracting
the fluid with a microhematocrit capillary tube.
Samples had to be pooled to get a sufficient volume to
measure osmolarity as above.
A fourth experiment tested the survival of limpets
transplanted lower in the intertidal. Two groups of
thirty limpets were transplanted from their home sites
to lower levels in the intertidal, chosen so limpets
could not move to higher levels. One group was trans¬
planted to a region that was submerged in three of the
four tide changes and not exposed to intense wave
action. A second group was moved to an exposed site
with very high wave action. A third group served as
controls and were moved to a different site but at the
same intertidal level they normally inhabit. Numbers
surviving were counted each day for 7 days.
Results
Figure One shows the data from the first of three
submergence experiments.
The control sample at time
zero was obtained during unusually high surf conditions
consistently wetting both control and experimental
limpets. Three hours after submergence, there was no
significant difference between tenacities and extra¬
corporeal fluid osmolarities among control and experi¬
mental limpets. There was a decrease in body fluid
osmolarity in the control groups between zero and three
hours coinciding with periods of heavy surf. The tena-
cities and extracorporeal fluid osmolarities in the
six- and nine-hour limpets were significantly differ-
ent, with p-values of less than 0.005 for random
sampling error. Twenty-nine hours after submergence,
the extracorporeal fluid osmolarity of the control
limpets was significantly greater than the experimental
group, with a p-value of less than 0.005. Total body
osmolarities of the control and experimental limpets
were similar at this time, which coincides with both
groups being splashed by high surf. There was no
significant difference in tenacities at twenty-nine
hours, but a significant difference in both extra-
corporeal fluid osmolarity and tenacity among the
control and experimental groups at forty-nine hours.
There was also a 50% decrease in tenacity of the
experimental group 49 hours after submergence, compared
with the initial control at time zero.
Figure Two shows the data from the second of three
submergence experiments. This experiment was conducted
under conditions of very warm weather and early low
followed by low-high tides. This maximized dessication
resulting in extracorporeal fluid and body fluid
osmolarities to 1300 mOsm. With this increase in
osmolarity, there was also an increase in tenacity from
the nine- to twenty-five-hour period. This relation-
ship is also seen from forty-nine to ninety-seven hours
in the control group. Prior to the forty-nine-hour
period, extracorporeal fluid and body fluid osmolari-
ties remained relatively constant in both the control
and experimental groups, as did the tenacities from
zero to six hours. Although it is unlikely that an
increased osmolarity is always indicative of an
increase in tenacity, the exposure to sea water
resulted in a significantly lower osmolarity and
tenacity in the experimental group with all p-values
less than 0.005 except in the nine-hour sample for
tenacity. Another important finding is that the
tenacities of the experimental limpets declined to
approximately 54% of the control value (at time zero)
after ninety-seven hours of submergence.
Figure Three shows the data from the final
submergence experiment in which all but the three-hour
experimental tenacities were significantly lower than
the control limpets with p-values less than 0.005. In
addition, the extracorporeal fluid osmolarities of the
experimental limpets were significantly lower than
controls at all sampling times. Body fluid osmolarities
followed extracorporeal fluid osmolarities in both
control and experimental groups. The ninety-seven-hour
experimental group showed a 64% decrease in tenacity
from the controls at time zero. There was also a rapid
drop in the tenacities of the experimental limpets from
the three- to nine-hour sampling times.
Figure Four shows the data from the effects of
transplanting experimental limpets to regions lower in
the intertidal. Compared with the control group, there
is significantly less survival in the exposed and non-
exposed limpets, with the exposed limpets losing 573 of
the original population and the non-exposed 47% of the
original number. In the control group, only one limpet
was lost during the seven-day period.
Discussion
In almost all cases, the experimental limpet
showed significantly lower osmolarities of extra-
corporeal fluid. Only during the three-hour period in
experiment one was this different statistically insig-
nificant and during this period, both control and
experimental limpets were getting heavily washed by
high surf. Correlating with the reduced osmolarities.
the tenacityies of the experimental limpets were all
lower than the controls with only four out of nineteen
determinations not statistically significant. In
experiment one the three- and twenty-five-hour samples
showed no significant difference; however, this coin-
cided with a period of heavy surf which was washing the
control limpets also. The nine-hour sample of experi¬
ment two and the three-hour sample of experiment three
show significant difference in tenacities due to high
variance and small sample size. Moreover, in the three
submergence experiments the ability of the limpets to
cling to the substrate declined rapidly in the first
nine hours and was less than 50% of the initial control
value by two days. This means that C. digitalis living
at lower levels may be placed in danger of being washed
off the rock, especially as their immersion time
increases as wave force increases. Though the safety
factor of a resting limpet is quite high (Denny et al.
1985), it is much lower for a crawling limpet and the
reducion in tenacity resulting from equilibration with
sea water may represent a significant physical stress
and play an important role in habitat selection in C.
digitalis. The transplant experiment designed to
"test" this demonstrated a 50% loss of limpet relocated
to lower levels over that of the controls. There could
be other reasons for this loss than being washed off
the rock (migration or predation); however, the most
rapid loss was in the exposed population, which is
consistent with the osmotic stress hypothesis. Also,
the work of Frank (1965) concerning seasonal upward
shifts in C. digitalis populations fits well with the
immersion effects on tenacity,
This study provides another view of the habitat
selection of C. digitalis. The initial study of Haven
(1971) described niche separation of C. digitalis and
C. scabra where C. digitalis had less resistance to
dessication but better competitive ability, thus
leading to its occupancy of vertical faces. Wolcott
(1973) rated them equivalent in dessication resistance
but indicated that dessication stress determined the
upper limit to residency. This does present a real
enigma for intertidal ecologists because it is the only
case where exposure to ambient sea water represents an
osmotic stress to a marine invertebrate. It is also
one of the few instances in which a physiological
stress is regulating the lower limits of distribution
of the adult population.
10
consistent with the osmotic stress hypothesis. Also
the work of Frank (1965) concerning seasonal upward
shifts in C. digitalis populations fits well with the
immersion effects on tenacity.
This study provides another view of the habitat
selection of C. digitalis. The initial study of Haven
(1971) described niche separation of C. digitalis and
C. scabra where C. digitalis had less resistance to
dessication but better competitive ability, thus
leading to its occupancy of vertical faces. Wolcott
(1973) rated them equivalent in dessication resistance
but indicated that dessication stress determined the
upper limit to residency. This does present a real
enigma for intertidal ecologists because it is the only
case where exposure to ambient sea water represents an
osmotic stress to a marine invertebrate. It is also
one of the few instances in which a physiological
stress is regulating the lower limits of distribution
of the adult population.
10
consistent with the osmotic stress hypothesis. Also,
the work of Frank (1965) concerning seasonal upward
shifts in C. digitalis populations fits well with the
immersion effects on tenacity.
This study provides another view of the habitat
selection of C. digitalis. The initial study of Haven
(1971) described niche separation of C. digitalis and
C. scabra where C. digitalis had less resistance to
dessication but better competitive ability, thus
leading to its occupancy of vertical faces.
Wolcott
(1973) rated them equivalent in dessication resistance
but indicated that dessication stress determined the
upper limit to residency. This does present a real
enigma for intertidal ecologists because it is the only
case where exposure to ambient sea water represents an
osmotic stress to a marine invertebrate. It is also
one of the few instances in which a physiological
stress is regulating the lower limits of distribution
of the adult population.
10
References
Baldwin, S. 1968. Manometric measurements of
respiratory activity in Acmaea digitalis and Acmaea
scabra. Veliger 11 (Suppl.): 79-82.
Breen, P. A. 1972. Seasonal migration and population
regulation in the limpet Acmaea (Collisella) digitalis.
Veliger 15: 133-141.
Denny, M. W. , T. L. Daniel, and M. A. R. Koehl. 1985.
Mechanical limits to size in wave-swept organisms.
Ecological Monographs 55: 69-102.
Frank, P. W. 1965. The biodemography of an intertidal
snail population. Ecology 46: 831-844.
Haven, S. B. 1971. Niche differences in the inter-
tidal limpets Acmaea scabra and Acmaea digitalis
(Gastropoda) in central California. Veliger 13:
231-248.
Wolcott, T. G. 1973. Physiological ecology and inter-
tidal zonation in limpets (Acmaea): a critical look at
"limiting factors." Biological Bulletin 145: 389-422.
11
Acknowledgements
I would like to thank Chuck Baxter, Tom Hahn and
John Kono for helping me to complete this project.
13
Figure One: Experiment 1
The osmolarities of extracorporeal fluid and body
fluid as a function of time in control and experimental
limpets.
Tenacity as a function of time in control and
experimental limpets.
Experiment +
200
I100
000
900
4.0
3.0
2.0
1.O

— Extracorporeal Fluid Osmo.
————- Body Fluid Osmo.
• Control
Experimenta
ta-

40
30
10
20
50
Time (hours)
• Control
Experimental

30
40
20
Time (hours
10
50
Figure Two: Experiment f2
The osmolarities of extracorporeal fluid and body
fluid as a function of time in control and experimental
limpets.
Tenacity as a function of time in control and
experimental limpets.
Experiment +2
1300
200
100
00
900
3.0
2.C
1.O
Extracorporeal Fluid Ösmo.
— Body Fluid Osmo.
• Control
n Experimenta!




d

V
20—49
0
73 97
Time (hours)
• Contro
Experimenta!
+ —1
1149 73—97
20
10
Time (hours)
Figure Three: Experiment f3
The osmolarities of extracorporeal fluid and body
fluid as a function of time in control and experimental
limpets.
Tenacity as a function of time in control and
experimental limpets.
Experiment +3
1400

21300
2
1200
100


000
900
0
4.0
3.0
2.0
1.O
10

— Extracorporeal Fluid Osmo.
————— Body Fluid Osmo.
• Control
Experimental

a
—— —

—
49 73—97
20
Time (hours)

S
• Contro
i Experimenta!

49
20
7397
Time (hours)
Figure Four
Number of limpets remaining as a function of time
for control, non-exposed and exposed limpets.
19
30
25
20
10
S
Control

8
8
Non-Exposed


Exposed
taataa-
Time (days)