26
INTRODUCTION
Habituation in marine worms is well documented. "Sedentaria or
tubiculous worms have been shown to habituate to tactile stimuli,
varying light intensities, and mechanical shock (Nicol, 1950). Detailed
studies on the nereid polychaetes (Clark, 1960, a, b) have shown
habituation to be a characteristic of "errantia" behavior. Habituation
of the withdrawal reflex has been viewed as a mechanism for resuming
normal behavior despite the presence of a continuous danger signal
(Nicol, 1950).
The cirratulid polychaete Cirriformia spirabrancha is found
beneath the surface of the sand in flexible tubes of substrate particles
embedded in a mucous matrix. The worm does not appear to possess discrete
photoreceptors. It is sensitive to sudden mechanical shock, and tactile
stimuli. The withdrawal reflex of C. spirabrancha to mechanical vibration
has been successfully habituated. Retention of habituation was shown to be
present 1 hour after initial habituation. When tested 6 hours after initial habituatior
there was no noticeable retention of the initial habituation. There is a great deal
of variation among individuals in the rate of habituation tovibrational stimuli.
MATERIALS AND METHODS
The organisms used in this experiment were collected in the inter¬
tidal mud under the Monterey Municipal pier at Monterey, Calif. on
April 22, 1968. The worms were individually cultured in petri dishes
which were submerged on a sea table and supplied with a constant flow
of aereated water. A layer of five to ten millimeters of black sand
from the natural habitat was placed in each petri dish to simulate
the natural environment. One group of worms was allowed to live at
least one month under laboratory conditions before it was used in
habituation experiments. As a control, a second group of worms was
collected and used in experiments within two days of collection.
In preparation for an experiment worms were carefully removed
from the sand with a blunt glass rod and transferred to an adjacent
empty petri dish. The worms were allowed to remain in the empty
plate long enough to shed the mucous-substrate burrow which surrounded
them. They were then transferred to a shallow cylindrical trough made
of hardened Polyform (see diagram). The trough was then placed on the
bottom of a finger bowl, a plastic petri plate was placed over the
trough to seal it from above and the bowl was then filled with sea water
to a depth sufficient to cover the petri plate and trough. Any trapped
bubbles were then released and the glass rod of the vibrator apparatus
(see diagram) was securely clamped down over the center of the trough.
The worm was allowed to remain undisturbed until its movements indicated
the resumption of normal activity (usually 5-15 min.). Stimuli were
then delivered with a duration of 1 second (40.5 sec.) with 1 second
(40.5 sec.) intervals between stimuli. Twenty-five consecutive trials
without a visible response were chosen as the criterion of habituation.
When the criterion was reached the worm was tested (by tactile stimulation)
for possible muscular fatigue, then it was returned to its petri dish
to await retesting after a precise time interval. Following the retest,
each worm was weighed and then discarded.
To avoid possible side effects of residual mucous (McConnell, 1966)
the trough was cleansed with sulfuric acid before each run and new
sand was placed in the bottom.
*Polyform is a pliable modeling material manufactured by the Polyform
Products Co., 9416 W. Irving Park Rd., Schiller Park, Ill. 60176.
Polyform bakes to a permanent hardness in 15 minutes at 300 F.
RESULTS
1. Response Characteristics.
There is a great deal of variability evident in the worm's
response to the stimulus. The degree, duration, and speed of contraction
is extremeley variable. The anterior region, particularly the prostomium,
appears to be unusually sensitive in comparison with the rest of the
body. This is particularly true when the worm has extended the anterior
portion of its body and is actively probing or searching the trough
with its snout. Mild tactile stimulation with a blunt glass rod of the
snout and prostomium in comparison with similar stimulation of mid and
hind parts of the animal support the observation that the prostomial
region is the most sensitive part of the body.
Contraction in response to the stimulus is easily differentiated
from normal body movements in that the contractions are initiated
during or immediately after the stimulus, are pronounced and rapid
in movement, and are almost always followed by a rapid recovery to the
original posture. The recovery period is slower than the contraction
but in most cases faster than the interval between stimuli. Normal
body movements are generally slow and deliberate with a tendency for
M.
the body to slowly contract toward the head in a moving animal.
2. Rate of Initial Habituation.
Ten laboratory worms took an average of 65.3 trials to reach
complete habituation. Standard deviation for the laboratory worms
was 14.1 trials. As a control, ten fresh worms were tested with
an average of 69.0 trials to reach criterion. Standard deviation for
the fresh worms was 4l.1 trials.
29
3. Retention of Habituation.
Five groups of worms were trained to criterion and then
retrained as follows:
GROUP A — Retrain after one hour
GROUP B — Retrain after six hours
GROUP C — Retrain after twelve hours
GROUP D — Retrain after eighteen hours
GROUP E — Retrain after twenty-four hours
The average rates and standard deviations of the above regime are:
Initial Ave. no. trials to Retest
Ave. no. trials to
Std. Dev. retest habituation Std. Dev.
initial habituation
GROUP A (10 worms)
42.1
69.1
36.7
15.2
GROUP B (10 worms)
57.8
28.4
42.0
15.4
69.7
GROUP C (10 worms)
29.3
41.6
59.3
62.9
60.0
GROUP D (9 worms)
45.8
43.3
64.5
33.8
50.6
38.4
GROUP E (9 worms)
(see diagrams 2, 3, 4, 5, 6)
DISCUSSION
Studies to investigate the possible correlation between size (wet weight) and
the rate of habituation have shown that the extreme variability of rate
cannot be attributed to size (see diagram.7). Similar studies show that
this variability cannot be attributed to tidal cycle or day-night cycle.
In some cases it is possible that experimental conditions such as residual
sulfuric acid may have contributed to the variability of a few organisms.
C. spirabrancha is highly thigmotactic. When worms are allowed to
remain in their mucous-substrate burrow they are less reactive. Shedding
of the burrow apparently stimulates the search for suitable substrate
as most organisms attempt to burrow in the small amount of sand on the
bottom of the trough. Even though the trough provides some measure of
security it is possible that the response of the worm to this strange
environment is abnormal. The difficulties of observations in the
natural sand environment make controls of this type infeasible thus
it must in some measure be assumed that behavior in the trough is not
distorted as compared to natural behavior.
Cirriformia spirabrancha appears to have no active predators
during the spring (Judd, 1968). The withdrawal reflex can be interpreted
not so much as a means of preventing predation but of maintaining itself
in the sand environment. Field observations show that a slight disturbance
of the sand above the worm will bring about immediate withdrawal of the
tentacles. Natural disturbances such as those brought about by the
rushing surf frequently move great volumes of the substrate. Self preservation
of Cirriformia would necessitate a means of detecting and then avoiding
an unstable environment above. Habituation to variable but innocuous
turbulence is also necessary to continue its normal functions.
When the data on retention of habituation åre subjected to the
standard t test a significant difference in the rate of habituation
at the 95% confidence interval for Group A is observed. This indicates
that there is a definite retention of the habituated response lasting
at least one hour after the worms vare initially tested. For the remaining
groups there there is no significant difference noted with thet test.
This indicates that retention of the habituated response had disappeared
during the subsequent time intervals.
36
ACKNOWLEDGEMENT
I would like to express my sincere thanks to the faculty and staff
of Hopkins Marine Station for their help in this study. An especial
thanks to Mr. Roger Szal whose aid and criticisms have helped me
throughout this academic quarter. This work was supported in part by
the Undergraduate Participation Program of the National Science
Foundation Grant GY-4369.
BIBLIOGRAPHY
1. Clark, R. B. (1960). Habituation of the polychaete Nereis to sudden
stimuli. I. General properties of the habituation process. Anim.
Behav., 8, 83-92.
2. Clark, R.B. (1960). Habituation of the polychaete Nereis to sudden
stimuli. II. Biological significance of habituation. Anim. Behav.,
8, 92-103.
3. Judd, T. (1968). Predation in Cirriformia spirabrancha. Unpublished
paper, Hopkins Marine Station.
L. McConnell, J.V. (1967)."Specific factors influencing planarian
behavior." Chemistry of Learning. Ed. by W.C. Corning and S.C.
Ratner. New York: Pleunum Press. 217-233.
5. Nicol, J. A. C. (1950). Responses of Branchiomma vesiculosum (Montagu)
to photic stimulation. J. mar. biol. Assoc. U.K., 29, 303-320.
3
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