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 ELECTRICAL LERDS 12 vost 95e VrE -Sem- lem END VIEU (PERFORATTo) 44co- Taoaen CONTROt sorred Leon Supro BARS Bete-Buz: GERSS RoD Perat drsn rsse Bee Teouca AePARATUS FOR VEBRRTTONAL HRBETURTTON Tor Vreu A 12-282 IHHE It H 20 Squares to the Inch + tttttttt f o 12-282 C 20 Squares to the Inch t 12-282 20 Squares to the Inch S H 29 ... 12-282 HHE He + 20 Squares to the Inch I t A. 12-282 HE 20 Squares to the Inch 12-282 t + 20 Squares to the Inch s H e t H H I T H ouf u 01 saenbg 0z