Abstract
The columellar muscle (foot-shell retractor) of the intertidal limpet
Collisella digitalis is responsible for the tenacity with which this limpet
can hold onto rock substrate in response to mechanical stress (i.e. wave
action and predation) (Fretter, 1962).
Consequently, dessication and splash provide great osmotic stress upon
this intertidal limpet. This paper describes the varying excitability and
"catch" characteristics of the foot-shell retractor muscle under varying
osmotic stresses.
INTRODUCTION
Causes of intertidal zonation for Collisella digitalis and other
Collisella (formerly Acmaea; Lindberg, 1981) of the Central California Coast
have been shown to result from physical as well as biological "limiting
factors." The upper limiting factors for C. digitalis are set by
dessication and temperature tolerances (Carefoot, 1977, Wolcott, 1973).
Lower limiting factors are traditionally thought to be set by biological
factors. These include competition for space and food resources (Haven,
S.B. 1973, 1971) as well as behavioral limitations (Wolcott, 1973). Thus,
physical limiting factors have often been cited in the literature for
limiting upper distribution but apparantly not so for the lower distribution
of C. digitalis. Many recognize this developing concept, that the upper
limits of distribution are physically determined and lower limits determined
by biological factors (Carefoot, 1973, Connell, 1961).
Physically determined lower limits of distribution, however, could
affect C. digitalis. Experiments reported in this paper were designed to
investigate the osmotic stress C. digitalis experiences during low tide
dessication and high tide submersion. The indices of stress used were
contractile properties of the shell-foot retractor muscle under varying
osmotic conditions. This muscle, also called the columellar muscle, is used
by the limpet to hold tenaciously onto rock substrates and must be largely
responsible for the animal's ability to live in exposed areas experiencing
great wave action. This muscle is also responsible for the shell clamp-down
behavior which provides protection from predators (Fretter, 1962).
MATERIALS AND METHODS
Specimens were collected from the intertidal zone at Cabrillo Point,
Pacific Grove, California, in the afternoon just before high tide to ensure
that the limpets had been subject to several hours of natural dessication
stress prior to study. Three groups were established. The first was placed
in sea water for five hour to induce isoosmotic conditions simulating
coverage by waves and splash during the high tide cycle. After the five
hour soak period the limpets were removed, and osmolalities of the
extra-corporeal fluid were measured by extraction of fluid with a 20 gauge
syringe and determination by a freezing-point depression osmometer. These
animals constituted the "full recovery" group. The foot-shell columellar
muscle was excised in sections of 3x 1 mm, with both ends intact, and
mounted for shortening experiments in which muscles were stimulated with
single, repetitive, electrical shocks. The electrode consisted of two 3x 4
pieces of platinum foil coated with platinum black. Electrodes were
positioned tranversely within 0.5 mm of the muscle. Electrode pulses were
provided with a Grass S44 stimulator. Shortening (under (1 gram of load)
was measured with a Phipps and Bird Displacement Transducer model ST-2 and
recorded with a Brush Recorder Mark 280.
For the second and third groups extra-corporeal fluid osmolality was
measured immediately after removal from intertidal sites. Muscles were
excised and studied in a manner similar to the first group. Note that
limpets in the second group were bathed in sea water only for the duration
of the experiment and were designated the "No Recovery" group.
The third group was bathed in hypertonic sea water (1300 mosm)
corresponding to the osmolalities of the animals in the field (1200-1400
mosm). Hypertonic sea water was prepared with 14.61 g Nacl in one liter sea
water then diluted with sea water until osmolality reached 1300 mosm. This
last group was termed the "Hyperosmotic" group.
Results
Threshold Twitch Behavior
Muscles from each group studied, "Full Recovery (Full R), No Recovery
(No R), and Hyperosmotic (Hyp)," respond to a single, brief (0.5 ms)
electrical shock (10 V) with a slow twitch. Time to peak was approximately
3-5 sec and time to half-relaxation 8-14 sec. Amplitudes of these near
threshold twitches are specific for the different groups of muscles.
Responses, measured inmm shortened, are compared in Figure 1 for single 10
V shock with durations varying from 0.5 to 2.0 msec. The Full R group
produced the largest twitches. Comparatively smaller twitches were recorded
from the No R group, and the smallest twitches from the Hyp group.
Figure 2 shows results of an analogous experiment employing 20 V
shocks. Twitches for all groups are larger than those elicited by 10 V
stimuli. Again the Full R group showed greater shortening response than the
other groups. With larger auochs, reugenses of the No R and Ryp groups were
not distinguishable from one another.
Figure 3 illustrates shortening recordings of the three groups during
tetanic stimulation with 20 V shocks at 200 Hz. Time to maximal shortening
ranged from 16 sec to 70 sec for the three groups. Muscles responded with
greatest peak shortening in Full R, but amplitudes were not as distinct from
No R and Hyp group amplitudes as amplitudes were in response to single
shocks. Time to peak did not differ. Relaxation times were very different
(see Figure 3). Results are supported by other experiments and provided in
summary Table 1. Table 1 shows the mean times for maximum shortening, time
to peak, time to half-relaxation and relative osmolarity measurements.
Mean times to half-relaxation are very significant and reveal great
disparity between the three groups. Muscles studied in the hypertonic
medium clearly relax from tetanic stimulation more slowly than muscles from
the other two groups.
Discussion
The differential responses to varying osmotic conditions may indicate a
physical limit on the lower distribution of C. digitalis. The reasoning
behind this idea will be discussed.
Single shock twitches revealed decreased contractility for muscles
subject to high osmotic conditions. Hyp muscles showed markedly lower
twitching in response to even the smallest stimuli, compared to No R and
Full R groups. Specifically No R muscles (prepared in a manner identical to
Hyp muscles (1300 mosm) only being bathed in sea water (970 mosm)) showed
decidedly higher sensitivity to single 10 V shocks.
Based upon these results a limpet's ability to forage and move
locations is likely to be reduced under dessication stress. At the same
time, under near isoosmotic conditions (to sea water) limpet muscle has a
greater ability to shorten. Applied to field situations, this suggests
greater movement or greater ability for movement under near isoosmotic
conditions such as found during high tide compared to low tide dessication.
From behavioral studies it is known that C. digitalis moves actively during
high tide. Consequently, decreased ability for muscle shortening induced by
hyperosmotic conditions tested in this study corresponds to clumping
behavior and decreased movement observed in the field during low tides. The
extent to which behavior is influenced by physiologically suppressed or
enhanced muscles is as yet undetermined and deserves additional study.
Tetanic Responses
Tetani were elicited at shock frequencies of 200 Hz. There were noted
differences between the groups. Maximum shortening responses revealed that
the Full R group shortened the most relative to Hyp group shortening, with
the No R Group shortening least of all. However, this pattern cannot be
regarded as unequivocal due to shortening response being dependent upon size
of limpet tested and due to variability with a small sample size (n-tetanic
- 2/group). Thus although the degree of muscle shortening may be
osmotically dependent, this report cannot conclusively determine this
relationship.
Mean times to peak were recorded during the period of 25 to 33 sec
beginning from time of stimulation. Averages of these data fail to show any
significant differences between the groups.
The greatest difference in the three groups lies in the muscles
ability to maintain the shortened state following tetanic stimulation. The
osmotically stressed group, Hyp, revealed a far greater ability to maintain
a sustained shortened position than either of the other two groups. This
difference amounted to an approximately sixteen-times slower rate of
re-extension than for the lowest osmotically stressed group, Full R.
Hore work is needed to clarify results concerning the time to peak and
maximum shortening response by increasing the number in each sample group.
Further, varying osmolality using a variety of agents in addition to NaCl
will better characterize the muscle. Although force was not measured in the
present study, it is tempting to speculate that the slow rate of
re-extension is analagous to the molluscan "catch" response, in which a
muscle can maintain force for a very long time in the absence of continued
stimulation (Twarog, 1967). Thus re-extension responses, following tetani
in the three groups studied may indicate that osmotic conditions are an
important factor in maintenance of the "catch" state. If these assumptions
are accepted, then limpets in the near isoosmotic condition (to sea water)
of being "Fully Recovered" show a much weaker ability to maintain long
catch" states. Conversely, dessicated animals would find it much easier to
maintain a catch state for long periods and to maintain a firm hold on a
rock.
These effects of osmolality on limpet muscle suggest that limiting
factors C. digitalis encounters in the field could influence distribution
limits. Not only can the limpet be restricted by upper physical boundaries
due to dessication and heat stress, but may also experience lower physical
boundaries in addition to the many biological boundaries. Intriguinaly.
this lower physical limit might involve physiological alterations such as
reduced ability to maintain muscular catch associated with body fluid
osmolalities (isoosmotic with sea water) under high tide conditions.
References
Carefoot, T.
Pacific seashores; a guide to
intertidal ecology. Seattle: University of
Washington Press, 1961.
Connell, J.H. (1961). The influence of
interspecific competition and other factors on
the distribution of the barnacle Chathamalus
stellatus. Ecology. 42:710-723.
Fretter, V. a Graham, A. British prosobranch
molluses; Their functional anatomy and
ecology. London:Ray Society, 1962.
Haven, S.B. (1971). Niche differences in the
intertidal limpets, Acmaea scabra and Acmaea
digitalis (Gastropoda) in central California.
Veliger. vol. 13, no. 3.
Haven, S.B (1973). Competition for food
between intertidal gastropods, Acmaea scabra
and Acmaea digitalis. Ecology.
54(1):143-151.
Lindberg, D.R. Acmaeidae: Gastropoda
mollusca. Pacific Grove, California: Boxwood
Press, 1981.
Iwarog, B.M. (1967). Factors influencing
contraction in catch in Nytilus smooth muscle,
J. Physiology. 192 (3):847-856.
Wolcott, T.G. (1973). Physiological ecology
in intertidal zonation in limpets (Acmaea): a
critical look at "limiting factors.
Biol.
Bull. 145.
Figure Legends
Figure 1. Excitability varies with
osmolality. Figure 1 represents displacement
(mm) versus duration (ms) at 10 V shocks.
Note the greater shortening excitability of
f-rec to n-rec and h-osm.
Figure 2. Excitability varies with
osmolality. Figure 2 represents displacement
(mm) versus duration (ms) at 20 V shocks. Note
the greater shortening of f-rec to n-rec and
h-osm.
Figure 3. Hyperosmolality enhances long catch
responses. Comparison of peak to
half-relaxation records for the three groups
(pulse times are specific for the individual
traces). Shortening measurements are mean
values of all members in the separate groups.
Note the great differences in half-relaxation
time for the varying osmolality groups (see
Table 1).
Table 1. Comparitive table of the mean values
of shortening responses and tetanic shortening
characteristics of each group compared with
mean osmolality values of extra-corporeal
fluid. Most important values are the Times to
Half-relaxation.
i
o

15
1
)(
O

8
i

5
88
MAXIMUM
SHORTENING
(mm)
TIME T
PEAK
(sec)
TIME TO
HALF
RELAXATION
OSMOLARITY
(mosm)
FULL RECOVERY
1.41 f .62
25.3 - 11.3
19.2 min.
f 11.97
956.3 + 6.75
NO RECOVERY HYPEROSMOTIC (1300mosm)
98 + .95
1.37
33.0 + 6.98
24.5
48.5 min.
307.9 min.
f 18.74
1400 + 29.14
1385