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Trial and error learning in paramecium: A replication
BEHAVIORAL BIOLOGY, 7, 873-880 (1972), Abstract No. K-59R
Trial and Error Learning in Paramecium: A replication I
THOMAS E. HANZEL 2 and WILLIAM B. RUCKER
Mankato State College
Single paramecia were sucked into a capillary tube and allowed to escape back into a drop of their culture. Escape speed increased in early trials and then stabilized, thus replicating French's 1940 results, and supporting his hypothesis of learning in paramecia. Further, neither intertrial interval nor habituation to the drop changed the rate at which escape improved, though habituation increased escape speed over all trials.
French reported in 1940 that paramecia sucked by capillary action into a glass tube escaped back into the culture drop more rapidly on succeeding trials. Immediately before and immediately after training, each subject was allowed 1 min of free swimming over a grid so that a measure of general activity could be taken. There was no change in the mean number of squares crossed and no correlation between activity scores and escape latencies which would appear to control for pseudoconditioning, unless specific to the tube. French concluded that the change in escape latencies could best be understood in terms of learning. The French study has been cited as "seemingly unequivocal evidence of trial-and-error learning in paramecia" ( K e l l o g g , 1958) and as supporting " a fairly safe conclusion that true learning has been demonstrated" (Thorpe, 1966). We found no recent citation in a search of the Science Citation Index through 1971 nor in papers listed in 1970 by Coming and V. Burg. Apparently no attempt has been made in 32 years to verify French's observation that escape improves with practice. While a number of controls 1This research was supported by a Grant to the second author from the Mankato State College Faculty Research Council. The authors thank Colin McDiarmid, John Zimmer, Joseph Huber, and Paul Brandon for advice and thank Susan Kliebenstein and Dennis Meyer for help in recording data. Requests for reprints should be sent to W. B. Rucker, Department of Psychology, Mankato State College, Mankato, Minnesota 56001. 2present address: Department of Psychology, Faribault State Hospital, Faribault, Minnesota 55021. 873 Copyright © 1972 by Academic Press, Inc. All rights of reproduction in any form reserved.
874
HANZELAND RUCKER
are necessary to establish that such behavior constitutes learning, the simplicity of the procedure suggests that such controls are obtainable. The present experiment replicated the basic observation and explores two factors which might be expected to interact with the acquisition of the escape response. The subjects used were Paramecium caudatum (General Biological Inc.). The paramecia were recultured at biweekly intervals in a medium consisting of one grain of boiled wheat/100 cc distilled water. The 40 subjects tested ranged from 0.18 to 0.22 mm in length. The animals were cultured and tested in the same room, the temperature of which was maintained at 26.5°C. Prior to each series of training trials, a small volume of culture medium was placed in a depression slide. The drop had a diameter of approximately 6.0 mm in the depression. A single paramecium was transferred from the culture to the drop by means of a bacteriological loop 2.0 mm in diameter. A training trial was begun by lowering a capillary tube to the surface of the drop so that the animal was sucked up the tube from the surface by capillary action. The tube, 4.6 mm in length, was cut from a 20 lambda Microcap (Drummond Scientific Co.) which had an inner diameter of 0.63 mm (the tube used by French was 4.6 mm long and had an inner diameter of 0.6 mm). The volume drawn up into the tube in each trial depended on capillary action, the diameter of the tube, and the medium. Therefore, the same volume (which we calculated to be 1.44 lambda) was drawn into the tube on each trial for all subjects. The ends of the tube were squared and finished by sanding with 600 grit 3M carborundum paper attached to a wheel on a high speed drill (Dremel Model 2). The tube was held by the observer in a small drafting pen at an angle of 70-75 ° to the slide. There was, therefore, no net increase in heating of the tube over trials due to holding it by the experimenter. This angle allowed unobstructed observation of the subject through a stereoscopic microscope (Nikon SMZ-2). Overhead fluorescent lighting provided the only light source. When the subject was captured by capillary action, the observer called time to a second experimenter, who then started a stopwatch. The tube was held steady at the surface of the slide until the animal escaped back into the drop and time was called again. Movement of the tube was minimized in our laboratory by: (a) a wooden hand support affixed to the base of the microscope; (b) bracing the fourth finger against the stage of the scope; and (c) bringing the bottom edge of the capillary tube down to the surface of the slide as the subject was caught. This last technique involved a very small movement of the tube from the surface of the medium to the surface of the slide. The angle of the tube was maintained at 70-75 ° . Plenty of space was allowed, therefore, at the end of the tube for subsequent escape even toward the back of the tube. Due to the constraints of the situation (i.e., need to see the animal, how the tube is
LEARNING IN PARAMECIA
875
held), the tube could not vary more than between an angle of 60-80 ° . The variation was actually even less than this and was produced mainly by the location of the animal at the time of capture-a presumably random event. While variations in the angle may have added to the error variance, they did not introduce bias. The second experimenter recorded the latency of the response and subsequently gave the countdown for the next intertrial interval. During the intertrial interval, the observer removed the liquid from the capillary tube with a water powered aspirator. The suction end of the pump was a short length of polyethylene tubing (Intramedic PE 200) attached to the stage of tile microscope and pointing upward and away from the testing drop. After every second trial, approximately 4.0 lambda of culture medium was added to the drop to compensate mainly for the volumes (1.44 lambda) removed by the capillary tube on each trial. Each subject was trained for 20 consecutive trials. Three paramecia did not escape from the tube on the first trial within 17 min. They were excluded from the experiment and replaced by other subjects. The experiment was a 2 × 2 factorial design with repeated measures. With respect to the first factor, the subjects were either tested immediately after their introduction into the testing drop or were allowed a 3-rain period of habituation before training began. (In French's study, the animals were observed for 1 min before training began.) As the second factor, two intertrial intervals were used, 15 sec (as used by French) and 30 sec. The ten subjects in each treatment were run consecutively. Treatment groups were run over a 2-week period in the following or.der: (1).15 sec intertrial interval and no habituation; (2)30 sec intertrial interval and habituation; (3) 15 sec intertrial interval and habituation; and (4) 30 sec intertrial interval and no habituation. The escape latencies were transformed to reciprocals (escape speed) in order to reduce heterogeneity of variance. Homogeneity of variance could not be rejected by an F-max test performed on the transformed scores from the first, sixth, and twentieth trials ( P > 0.05; F-max = 2.01; 3 variances; dr= 40.). Statistical tests were provided by Myers (1966). Table 1 shows the analysis of variance over all 20 trials and an ad hoc analysis over trials 10-20. The increase in escape speed is quite reliable ( P < 0.005) in the overall analysis, but is essentially complete by the tenth trial, since the trials effect is not significant in the analysis of the last trials alone. The median escape latencies in this experiment are compared in Fig. 1 with those reported by French (1940). The more rapid escape by paramecia in the present experiment may be attributed in part to the effect of habituation which significantly speeded responses on all trials (P < 0.025). The habituation effect is shown in Fig. 2 in terms of speed, since the reciprocal of latency was used in the analysis. An effect of intertrial interval on escape speed was not evident in the over-all analysis of variance. That this effect reached significance ( P < 0.05) in the analysis of trial 10-20 is apparently due to a decrease
HANZEL AND RUCKER
876
TABLE 1 Analysis of Variance
Source of variation
Trials 1-20
df
Trials 10-20
df
Intertrial interval
1
M.S. .27144
F 2.58
Habituation
1
.65096
ITI × habituation
1
.00362
Error (a)
36
.10496
Trials
19
.05556
3.15 d
ITI × trials
19
.02497
1.41
10
.02411 1.37
Habituation× trials
19
.01494
.84
10
.01581
.90
ITI× Habituation× trials
19
.01650
.93
10
.01492
.85
684
.07161
360
.01752
Error (b)
1
M.S. F .33397 5.10a
6.20b
1
.62808 9.59 c
.03
1
.00344
36
.06546
10
0.1227
.05
.70
ap < 0,05. bp < 0.025. cp < 0.005. dp < 0.001. in intersubject variability in these later trials. Subjects tested at 30-sec intertrial intervals escaped slightly more rapidly than those tested at 15-sec intervals. Neither h a b i t u a t i o n n o r intertrial interval changed the rate at which escape speed improved reliably. Qualitatively, the paramecia in this" e x p e r i m e n t generally performed in a m a n n e r similar to that described b y French. During early trials, the subjects 30
25
0 20
,,z
,~
FRENCH (. ~'20)
r"x, z '~
5 In= 401 I
5
lb
I
15
2'0
TRIAL NUMBER
Fig. 1. Comparison of median escape latencies over training in present Experiment and in French (1940).
877
LEARNING IN PARAMECIA .3O 3. M I N . H A B I T U A T I O N
,o:y
m~.25 c
.~ .20
J/
~3
m
I
/I
A d/
a/v"
.
/ /\ '¢ /,W' ,"2. 2~¢B'~uATl°" I ,,l (n=20)
,, / L-,
l I lI 1
5
10 15 TRIAL NUMBER
20
Fig. 2. Effect of habituation on escape speed.
would swim short distances up and down the tube, beating back and forth at each level. On later trials, the paramecia beat back and forth only a few times, and then took a single dive down and out of the tube. In addition, some of the animals in the present experiment appeared to take the surface of the fluid in the tube as a cue to begin the long dive, an observation not recorded by French. In both experiments, individual behavior varied considerably from trial to trial. French (1940) reported that approximately half of his subjects showed no evidence of learning and continued to make relatively delayed escape throughout training. Approximately a quarter of the individuals in the present experiment failed to demonstrate improved escape speed, but these animals began the experiment with relatively rapid escape speed. In French's experiment, no mention was made of the number of subjects drawn only part way up the capillary tube. Since the subjects might be expected to escape the shorter distance in less time on these trials, such occurrences were, carefully tallied in a related study (Hanzel and Rucker, 1972). This occurred 35% of the time. In this study, there were neither differences between treatment groups n o r differences over trials for the number of occasions when the subjects were not sucked to the top. Thus, the increase in escape speed over trials is not likely to be related to this source of variability. In this experiment, we also measured activity before and after
878
HANZEL AND RUCKER
training, but with a grid finer than French's. In this study, we picked up an increase in activity which French missed. There was, however, n o statistical relation between change in activity and change in escape speed. In another study (Huber, Rucker, and McDiarmid, 1972), we have measured activity between trials and found again what may be a change in activity independent of the change in escape speed. It also seems unlikely that the minor disturbance o f adding fluid to the drop while training was in progress contributed to the change in escape speed. French (1940) used a much larger drop and did not need to add any fluid, yet both his and our study produced reliable increases in early training trials. In summary, the present experiment systematically replicates the finding that with practice, paramecia escape more rapidly from a capillary tube (French, 1940; cf. Day and Bentley, 1911; Smith, 1908). The argument that this modification of behavior may be due to learnirtg is like the statistical argument that the alternative hypothesis should be accepted because the null hypothesis is improbable. Nonlearning explanations fall into two categories, those based on progressive changes in the external environment of the subject and those based on internal changes. The external test environment includes both the drop and the tube. The conditions in the drop were maintained reasonably constant during the experiment. More importantly, time spent by the subjects in the drop, either before training (habituation) or during training (intertrial interval) had no effect on the rate at which escape behavior was acquired. It should be noted that in the factorial design used, all 40 subjects were used in reaching each of these conclusions. It could be argued that changes in the tube environment produced the behavioral modification. If so, the effect would have to have been a transient one since animals later tested in the same tube had no advantage in initial escape speed. A transient effect might be produced by a build-up of carbonic acid in the tube, perhaps producing more supple animals (Buytendijk, 1919; Jensen, 1957). This factor appears irrelevant, however, because the same behavioral change is reported in the present experiment, wherein the tube was air-dried between trials, as in French's study in which CO2-1aden breath was used to force the water out (French, 1972). Other progressive changes in the external environment, such as a build-up of some nonvolatile chemical released by the paramecia, or a change in the physical stimulus properties in the tube were controlled in the following manner. Subjects (N= 15) were individually sucked into the capillary tube which had been sealed into a drop dispenser, and for the first nine trials (ITI of 15 in.) the tube was removed from the drop to prevent the subject from swimming back. Instead, the drop dispenser was used to blow the subject gently back into the test drop after he had spent the same period of time in the tube as the median escape latency for that trial number in the experiment reported above (see Fig. 2). On trials 10-19 each subject was allowed to escape freely.
LEARNING IN PARAMECIA
879
Escape speed rose from 0.06 on the tenth trial to 0.10 on the nineteenth trial, though the difference was not significant. An additional 18 subjects which were tested under the same conditions had escape speeds which rose 0.10 on the tenth trial to 0.i9 on the nineteenth trial, again not significant. In both groups, escape speeds were substantially slower than those shown in Fig. 2. Thus, instead of producing conditions favoring rapid escape, passive movement of the subjects in and out of the tube actually interfered with the development of rapid escape. This result is consistent with the finding that animals maintained in the tube for 3 min prior to training escape slowly at first even though they subsequently improve in escape speed (Hanzel and Rucker, 1972). Internal changes in the subject which might explain their more rapid escape include: 1. fatigue (which would be expected to slow, rather than hasten, their escape); 2. adaptation (an argument raised by Buytendijk [1919], but weakened by the control experiments reported before); 3. pseudoconditioning (which would involve a direct correlation between the increase in escape speed and general activity and this has not been reported either by French [1940] or Hanzel and Rucker [1972]). The French procedure produces reliable changes in behavior of paramecia and allows a number of experimental variables to be explored with relative ease. At present, explanations of the behavioral change must still include the possibility of learning and, indeed, memory. In a subsequent study (Huber, Rucker, and McDiarmid, 1972), we gave 10 trials of training and then tested 27 subjects at each of four retention intervals (0, 6, 30, and 150 min). Rapid escape was maintained even after 150 min, which is considerably longer than the "memory time" reported in some protozoan studies.
REFERENCES Buytendijk, F. J. (1919). Acquisition d'habitudes par des etres uniceUulaires.Arch. Neerl. Physiol. 3, 455-468. Corning, W. C., and Von Burg, R. (1970). Protozoan learning-a bibliography J. BioL Psychol. 12, 91-96. Day, I. M. and Bentley, M. (1911). Note on learning in paramecium, J. Animal Behav. 1, 67-73. French, J. W. (1971). Personal communication. French, J. W. (1940). Trial and error learning in paramecium. J. Exp. Psychol. 26, 609-613. Hanzel, T.E., and Rucker, W. B. (1972). Escape training in paramecia. J. Biol. Psychol. (in press).
880
HANZELAND RUCKER
Huber, J. C., Rucker, W. B., and McDiarmid, C. G. (1972). Retention of escape training and activity changes in single paramecia (in preparation). Jensen, D. D. (1957). More on "learning" in paramecia. Science 1957, 126, 1341-1342. Kellogg, W. N. (1958). Worms, dogs, and paramecia. Science 127, 766-767. Myers, J. L. (1966). "Fundamentals of Experimental Design." Boston: AUyn and Bacon. Smith, S. (1908). The limits of educability in paramecium. Z Camp. Neurol. 18, 499-510. Thorpe, W. H. (1966). "Learning and Instinct in Animals," 2nd Ed., p. 189. Cambridge: Harvard University Press.
Trial and Error Learning in Paramecium: A replication I
THOMAS E. HANZEL 2 and WILLIAM B. RUCKER
Mankato State College
Single paramecia were sucked into a capillary tube and allowed to escape back into a drop of their culture. Escape speed increased in early trials and then stabilized, thus replicating French's 1940 results, and supporting his hypothesis of learning in paramecia. Further, neither intertrial interval nor habituation to the drop changed the rate at which escape improved, though habituation increased escape speed over all trials.
French reported in 1940 that paramecia sucked by capillary action into a glass tube escaped back into the culture drop more rapidly on succeeding trials. Immediately before and immediately after training, each subject was allowed 1 min of free swimming over a grid so that a measure of general activity could be taken. There was no change in the mean number of squares crossed and no correlation between activity scores and escape latencies which would appear to control for pseudoconditioning, unless specific to the tube. French concluded that the change in escape latencies could best be understood in terms of learning. The French study has been cited as "seemingly unequivocal evidence of trial-and-error learning in paramecia" ( K e l l o g g , 1958) and as supporting " a fairly safe conclusion that true learning has been demonstrated" (Thorpe, 1966). We found no recent citation in a search of the Science Citation Index through 1971 nor in papers listed in 1970 by Coming and V. Burg. Apparently no attempt has been made in 32 years to verify French's observation that escape improves with practice. While a number of controls 1This research was supported by a Grant to the second author from the Mankato State College Faculty Research Council. The authors thank Colin McDiarmid, John Zimmer, Joseph Huber, and Paul Brandon for advice and thank Susan Kliebenstein and Dennis Meyer for help in recording data. Requests for reprints should be sent to W. B. Rucker, Department of Psychology, Mankato State College, Mankato, Minnesota 56001. 2present address: Department of Psychology, Faribault State Hospital, Faribault, Minnesota 55021. 873 Copyright © 1972 by Academic Press, Inc. All rights of reproduction in any form reserved.
874
HANZELAND RUCKER
are necessary to establish that such behavior constitutes learning, the simplicity of the procedure suggests that such controls are obtainable. The present experiment replicated the basic observation and explores two factors which might be expected to interact with the acquisition of the escape response. The subjects used were Paramecium caudatum (General Biological Inc.). The paramecia were recultured at biweekly intervals in a medium consisting of one grain of boiled wheat/100 cc distilled water. The 40 subjects tested ranged from 0.18 to 0.22 mm in length. The animals were cultured and tested in the same room, the temperature of which was maintained at 26.5°C. Prior to each series of training trials, a small volume of culture medium was placed in a depression slide. The drop had a diameter of approximately 6.0 mm in the depression. A single paramecium was transferred from the culture to the drop by means of a bacteriological loop 2.0 mm in diameter. A training trial was begun by lowering a capillary tube to the surface of the drop so that the animal was sucked up the tube from the surface by capillary action. The tube, 4.6 mm in length, was cut from a 20 lambda Microcap (Drummond Scientific Co.) which had an inner diameter of 0.63 mm (the tube used by French was 4.6 mm long and had an inner diameter of 0.6 mm). The volume drawn up into the tube in each trial depended on capillary action, the diameter of the tube, and the medium. Therefore, the same volume (which we calculated to be 1.44 lambda) was drawn into the tube on each trial for all subjects. The ends of the tube were squared and finished by sanding with 600 grit 3M carborundum paper attached to a wheel on a high speed drill (Dremel Model 2). The tube was held by the observer in a small drafting pen at an angle of 70-75 ° to the slide. There was, therefore, no net increase in heating of the tube over trials due to holding it by the experimenter. This angle allowed unobstructed observation of the subject through a stereoscopic microscope (Nikon SMZ-2). Overhead fluorescent lighting provided the only light source. When the subject was captured by capillary action, the observer called time to a second experimenter, who then started a stopwatch. The tube was held steady at the surface of the slide until the animal escaped back into the drop and time was called again. Movement of the tube was minimized in our laboratory by: (a) a wooden hand support affixed to the base of the microscope; (b) bracing the fourth finger against the stage of the scope; and (c) bringing the bottom edge of the capillary tube down to the surface of the slide as the subject was caught. This last technique involved a very small movement of the tube from the surface of the medium to the surface of the slide. The angle of the tube was maintained at 70-75 ° . Plenty of space was allowed, therefore, at the end of the tube for subsequent escape even toward the back of the tube. Due to the constraints of the situation (i.e., need to see the animal, how the tube is
LEARNING IN PARAMECIA
875
held), the tube could not vary more than between an angle of 60-80 ° . The variation was actually even less than this and was produced mainly by the location of the animal at the time of capture-a presumably random event. While variations in the angle may have added to the error variance, they did not introduce bias. The second experimenter recorded the latency of the response and subsequently gave the countdown for the next intertrial interval. During the intertrial interval, the observer removed the liquid from the capillary tube with a water powered aspirator. The suction end of the pump was a short length of polyethylene tubing (Intramedic PE 200) attached to the stage of tile microscope and pointing upward and away from the testing drop. After every second trial, approximately 4.0 lambda of culture medium was added to the drop to compensate mainly for the volumes (1.44 lambda) removed by the capillary tube on each trial. Each subject was trained for 20 consecutive trials. Three paramecia did not escape from the tube on the first trial within 17 min. They were excluded from the experiment and replaced by other subjects. The experiment was a 2 × 2 factorial design with repeated measures. With respect to the first factor, the subjects were either tested immediately after their introduction into the testing drop or were allowed a 3-rain period of habituation before training began. (In French's study, the animals were observed for 1 min before training began.) As the second factor, two intertrial intervals were used, 15 sec (as used by French) and 30 sec. The ten subjects in each treatment were run consecutively. Treatment groups were run over a 2-week period in the following or.der: (1).15 sec intertrial interval and no habituation; (2)30 sec intertrial interval and habituation; (3) 15 sec intertrial interval and habituation; and (4) 30 sec intertrial interval and no habituation. The escape latencies were transformed to reciprocals (escape speed) in order to reduce heterogeneity of variance. Homogeneity of variance could not be rejected by an F-max test performed on the transformed scores from the first, sixth, and twentieth trials ( P > 0.05; F-max = 2.01; 3 variances; dr= 40.). Statistical tests were provided by Myers (1966). Table 1 shows the analysis of variance over all 20 trials and an ad hoc analysis over trials 10-20. The increase in escape speed is quite reliable ( P < 0.005) in the overall analysis, but is essentially complete by the tenth trial, since the trials effect is not significant in the analysis of the last trials alone. The median escape latencies in this experiment are compared in Fig. 1 with those reported by French (1940). The more rapid escape by paramecia in the present experiment may be attributed in part to the effect of habituation which significantly speeded responses on all trials (P < 0.025). The habituation effect is shown in Fig. 2 in terms of speed, since the reciprocal of latency was used in the analysis. An effect of intertrial interval on escape speed was not evident in the over-all analysis of variance. That this effect reached significance ( P < 0.05) in the analysis of trial 10-20 is apparently due to a decrease
HANZEL AND RUCKER
876
TABLE 1 Analysis of Variance
Source of variation
Trials 1-20
df
Trials 10-20
df
Intertrial interval
1
M.S. .27144
F 2.58
Habituation
1
.65096
ITI × habituation
1
.00362
Error (a)
36
.10496
Trials
19
.05556
3.15 d
ITI × trials
19
.02497
1.41
10
.02411 1.37
Habituation× trials
19
.01494
.84
10
.01581
.90
ITI× Habituation× trials
19
.01650
.93
10
.01492
.85
684
.07161
360
.01752
Error (b)
1
M.S. F .33397 5.10a
6.20b
1
.62808 9.59 c
.03
1
.00344
36
.06546
10
0.1227
.05
.70
ap < 0,05. bp < 0.025. cp < 0.005. dp < 0.001. in intersubject variability in these later trials. Subjects tested at 30-sec intertrial intervals escaped slightly more rapidly than those tested at 15-sec intervals. Neither h a b i t u a t i o n n o r intertrial interval changed the rate at which escape speed improved reliably. Qualitatively, the paramecia in this" e x p e r i m e n t generally performed in a m a n n e r similar to that described b y French. During early trials, the subjects 30
25
0 20
,,z
,~
FRENCH (. ~'20)
r"x, z '~
5 In= 401 I
5
lb
I
15
2'0
TRIAL NUMBER
Fig. 1. Comparison of median escape latencies over training in present Experiment and in French (1940).
877
LEARNING IN PARAMECIA .3O 3. M I N . H A B I T U A T I O N
,o:y
m~.25 c
.~ .20
J/
~3
m
I
/I
A d/
a/v"
.
/ /\ '¢ /,W' ,"2. 2~¢B'~uATl°" I ,,l (n=20)
,, / L-,
l I lI 1
5
10 15 TRIAL NUMBER
20
Fig. 2. Effect of habituation on escape speed.
would swim short distances up and down the tube, beating back and forth at each level. On later trials, the paramecia beat back and forth only a few times, and then took a single dive down and out of the tube. In addition, some of the animals in the present experiment appeared to take the surface of the fluid in the tube as a cue to begin the long dive, an observation not recorded by French. In both experiments, individual behavior varied considerably from trial to trial. French (1940) reported that approximately half of his subjects showed no evidence of learning and continued to make relatively delayed escape throughout training. Approximately a quarter of the individuals in the present experiment failed to demonstrate improved escape speed, but these animals began the experiment with relatively rapid escape speed. In French's experiment, no mention was made of the number of subjects drawn only part way up the capillary tube. Since the subjects might be expected to escape the shorter distance in less time on these trials, such occurrences were, carefully tallied in a related study (Hanzel and Rucker, 1972). This occurred 35% of the time. In this study, there were neither differences between treatment groups n o r differences over trials for the number of occasions when the subjects were not sucked to the top. Thus, the increase in escape speed over trials is not likely to be related to this source of variability. In this experiment, we also measured activity before and after
878
HANZEL AND RUCKER
training, but with a grid finer than French's. In this study, we picked up an increase in activity which French missed. There was, however, n o statistical relation between change in activity and change in escape speed. In another study (Huber, Rucker, and McDiarmid, 1972), we have measured activity between trials and found again what may be a change in activity independent of the change in escape speed. It also seems unlikely that the minor disturbance o f adding fluid to the drop while training was in progress contributed to the change in escape speed. French (1940) used a much larger drop and did not need to add any fluid, yet both his and our study produced reliable increases in early training trials. In summary, the present experiment systematically replicates the finding that with practice, paramecia escape more rapidly from a capillary tube (French, 1940; cf. Day and Bentley, 1911; Smith, 1908). The argument that this modification of behavior may be due to learnirtg is like the statistical argument that the alternative hypothesis should be accepted because the null hypothesis is improbable. Nonlearning explanations fall into two categories, those based on progressive changes in the external environment of the subject and those based on internal changes. The external test environment includes both the drop and the tube. The conditions in the drop were maintained reasonably constant during the experiment. More importantly, time spent by the subjects in the drop, either before training (habituation) or during training (intertrial interval) had no effect on the rate at which escape behavior was acquired. It should be noted that in the factorial design used, all 40 subjects were used in reaching each of these conclusions. It could be argued that changes in the tube environment produced the behavioral modification. If so, the effect would have to have been a transient one since animals later tested in the same tube had no advantage in initial escape speed. A transient effect might be produced by a build-up of carbonic acid in the tube, perhaps producing more supple animals (Buytendijk, 1919; Jensen, 1957). This factor appears irrelevant, however, because the same behavioral change is reported in the present experiment, wherein the tube was air-dried between trials, as in French's study in which CO2-1aden breath was used to force the water out (French, 1972). Other progressive changes in the external environment, such as a build-up of some nonvolatile chemical released by the paramecia, or a change in the physical stimulus properties in the tube were controlled in the following manner. Subjects (N= 15) were individually sucked into the capillary tube which had been sealed into a drop dispenser, and for the first nine trials (ITI of 15 in.) the tube was removed from the drop to prevent the subject from swimming back. Instead, the drop dispenser was used to blow the subject gently back into the test drop after he had spent the same period of time in the tube as the median escape latency for that trial number in the experiment reported above (see Fig. 2). On trials 10-19 each subject was allowed to escape freely.
LEARNING IN PARAMECIA
879
Escape speed rose from 0.06 on the tenth trial to 0.10 on the nineteenth trial, though the difference was not significant. An additional 18 subjects which were tested under the same conditions had escape speeds which rose 0.10 on the tenth trial to 0.i9 on the nineteenth trial, again not significant. In both groups, escape speeds were substantially slower than those shown in Fig. 2. Thus, instead of producing conditions favoring rapid escape, passive movement of the subjects in and out of the tube actually interfered with the development of rapid escape. This result is consistent with the finding that animals maintained in the tube for 3 min prior to training escape slowly at first even though they subsequently improve in escape speed (Hanzel and Rucker, 1972). Internal changes in the subject which might explain their more rapid escape include: 1. fatigue (which would be expected to slow, rather than hasten, their escape); 2. adaptation (an argument raised by Buytendijk [1919], but weakened by the control experiments reported before); 3. pseudoconditioning (which would involve a direct correlation between the increase in escape speed and general activity and this has not been reported either by French [1940] or Hanzel and Rucker [1972]). The French procedure produces reliable changes in behavior of paramecia and allows a number of experimental variables to be explored with relative ease. At present, explanations of the behavioral change must still include the possibility of learning and, indeed, memory. In a subsequent study (Huber, Rucker, and McDiarmid, 1972), we gave 10 trials of training and then tested 27 subjects at each of four retention intervals (0, 6, 30, and 150 min). Rapid escape was maintained even after 150 min, which is considerably longer than the "memory time" reported in some protozoan studies.
REFERENCES Buytendijk, F. J. (1919). Acquisition d'habitudes par des etres uniceUulaires.Arch. Neerl. Physiol. 3, 455-468. Corning, W. C., and Von Burg, R. (1970). Protozoan learning-a bibliography J. BioL Psychol. 12, 91-96. Day, I. M. and Bentley, M. (1911). Note on learning in paramecium, J. Animal Behav. 1, 67-73. French, J. W. (1971). Personal communication. French, J. W. (1940). Trial and error learning in paramecium. J. Exp. Psychol. 26, 609-613. Hanzel, T.E., and Rucker, W. B. (1972). Escape training in paramecia. J. Biol. Psychol. (in press).
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Huber, J. C., Rucker, W. B., and McDiarmid, C. G. (1972). Retention of escape training and activity changes in single paramecia (in preparation). Jensen, D. D. (1957). More on "learning" in paramecia. Science 1957, 126, 1341-1342. Kellogg, W. N. (1958). Worms, dogs, and paramecia. Science 127, 766-767. Myers, J. L. (1966). "Fundamentals of Experimental Design." Boston: AUyn and Bacon. Smith, S. (1908). The limits of educability in paramecium. Z Camp. Neurol. 18, 499-510. Thorpe, W. H. (1966). "Learning and Instinct in Animals," 2nd Ed., p. 189. Cambridge: Harvard University Press.