The Effect of Induced Cannibalism on Learning in Planaria
Victor Mircea
TJHSST
Biology I, Period 6
Dr. Wood
March 27, 2004
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Contents
Introduction ……………………………………………………………….. 3
Literature Review …………………………………………………. 3
Planaria ……………………………………………………. 3
Generational cannibalism …………………………………. 5
Learning …………………………………………………… 7
Experimental Design ……………………………………………… 8
Materials and Methods ...……………………………………………………8
Technology Component ……………………………………………. 8
Apparatus …………………………………………………... 8
Operation …………………………………………………… 9
Procedures and Materials …………………………………………... 9
Results ……………………………………………………………………… 10
Data …………………………………………………………………. 10
Statistics …………………………………………………………….. 11
Graphs ………………………………………………………………. 11
Discussion …………………………………………………………………... 11
Analysis …………………………………………………………….. 11
Summary ……………………………………………………………. 12
Bibliography ………………………………………………………………… 14
Appendix ……………………………………………………………………. 15
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Introduction
The purpose of this experiment was to find the effect of generational cannibalism
on the transfer of learning in planaria.
Transfer of intelligence through RNAi is an important concept, because RNAi can
modify key genes and DNA to change traits, such as memory and intelligence.
The hypothesis of this experiment was that if the generation of the planaria increases,
then the time to respond to stimuli would decrease. This response to stimuli is a form of simple
intelligence. The null hypothesis of this experiment was that if the generation of the planaria
increases, then the time to respond to stimuli would remain constant.
Literature Review
Planaria.
Planarian is a common name for several genera of the free-living (turbellarian)
flatworms belonging to the order Tricladida, a name that comes from their characteristic threebranched digestive cavities. Most species of planarians range from 1/8 inches to about 1
inches in length (.32–2.54 cm) although some giant tropical forms range up to 2 feet (60 cm)
(McConnell, 1966). The different species are white, gray, brown, or black; a few forms are
transparent. Many are striped or streaked and some are brightly colored. Although planarians
exist in marine or moist terrestrial habitats, most inhabit freshwater areas. Planarians in the
wild often eat small snails and other small herbivores. They crawl about over a trail of mucus
that they secrete by specialized epidermal cells; the smaller forms move about by means of
cilia on their ventral, or lower, surface, and larger species utilize muscular contractions as
well. Tactile and chemoreceptive cells (react to certain chemicals located in the environment),
located in the epidermis, serve as general sense organs. In many species, these cells clump in
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lobes at the sides of the head (Brown, Dustman, & Beck, 1965). Most planarians are also light
sensitive and in some, pigmented light-sensitive cells clump in two cups that serve as
primitive eyes. Planarians are usually either carnivorous or scavengers, depending on the
species. Certain types of planarians are cannibalistic, and thus they will attempt to ingest any
other small creatures that are around them, including other planarians. The mouth is located
near the middle of the ventral, or lower surface. Planarians are hermaphroditic; each individual
worm contains both male and female organs, and, most commonly, they reproduce sexually
(Best, 1960). However, species similar to the half-inch long (1.27-cm) Dugesia tigrina, which
is the most common planarian in the United States, are studied in classrooms and laboratories
for their additional capacity to reproduce asexually by transverse rupture of the body. A
rupture line develops behind the mouth, and while the back half of the worm anchors, the front
half moves forward until the worm snaps in half. Each half regenerates the missing parts. Such
planarians can also regenerate parts that they lose from their bodies (Brown, Dustman, &
Beck, 1965). There have been several studies done on the subject of planarian intelligence that
increases because of the cannibalization of other planaria that have already mastered a skill.
The trend from these studies makes it seem that intelligence transfers. The fact that planaria
possess this skill has many great implications. If humans could imitate this process somehow,
intelligence would transfer simply by ingesting learning pills (McConnell, 1966). Another odd
thing about planarians is that, when cut in certain places, they will reproduce incorrectly,
forming many interesting mutations. These can include, but are not limited to, having multiple
heads, having almost an entire planarian budding off one another, and having multiple tails.
Theoretically, it is possible to have a planarian with 20 tails and 20 heads, but it would
probably pull some extra heads and tails off before it reaches that milestone.
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Generational Cannibalism.
The independent variable of this experiment is generation cannibalism. In the 1960s, one
of the most talked about areas of neuroscience-concerned reports that chemical extracts, isolated
from animals that had been subjected to classical conditioning paradigms, could enhance
learning when transferred to their naïve counterparts (Smalheiser, Manev, & Costa, 2001). A
prominent worker in this field, James V. McConnell (1966), established that planarians
(flatworms) reliably turn in response to light or vibration. Taking advantage of the regenerative
capacity of planarians, he separated the head (containing the brain) from the tail in trained
animals, and reported that persistent behavioral changes occurred in animals that regenerated
from either half.
In their report, Brown, Dustman, and Beck (1965) talked about how planaria trained
under conditions that resulted in little or no learning in other animals (temporally separated light
and shock) and with different levels of conditioned stimulus luminance. Temporally separated
light and shock resulted in increased responsiveness that resembled altered behavior of planaria
trained with simultaneous light and shock. Increased luminance also caused increased
responsiveness. The data indicated that shock sensitizes planaria to light and causes spontaneous
division of the animals, resulting in shorter worms that are more responsive. The combined
effects of these variables resulted in increased response levels that others attribute to learning.
McConnell (1966) published an article in the journal Nature that reported results of
classical conditioning in brown planaria (Dugesia tigrina). In his study, he paired an electrical
shock (conditioning stimuli) with a flash of light (unconditioned stimuli). When the shock would
occur, the worms would contract and turn at the anterior end. The experiment’s control was a
group that received no electric shock, and a group that received light and shock at random. After
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training, the planaria received stimulus by light alone without electric shock. The planaria
exhibited the same type of response originally caused by the electric shock. McConnell followed
these tests with extinction trials that is they "reverse trained" the planaria to forget their earlier
learning.
Best (1960) suggested that perhaps "memory" is everywhere, in every cell, as it were,
dispersed throughout the whole organism. It is true that scientists believe memory is stored and
transferred via RNA, but perhaps memory is contained in every living cell.
RNAi occurs when double-stranded RNA that is expressed within, or taken up by, cells,
is cleaved into smaller protein-bound fragments that hybridize to endogenous cellular sequences,
resulting in selective degradation of specific endogenous mRNAs and the consequent
suppression of individual gene functions. The RNAi describes dozens of different genes in a
variety of invertebrate phyla, including planaria. Of the amazing features of RNAi that attract
attention, it is relevant here to note that one can induce RNAi by simply injecting doublestranded RNA into the body cavity in vivo, or even by feeding animals with bacteria that express
exogenous RNAs. According to Smalheiser, Manev, and Costa (2001), RNAi is something that
occurs in planaria, and it can transfer memory, as well as knowledge in these organisms.
Planaria may perform certain actions by using stimuli, under training. However, the
details and complexities of these mechanisms are not explicit to any degree of precision. All the
evidence seems to point to the fact that every cell holds memory (McConnell, 1966). This is an
important topic because, as Smalheiser, Manev, and Costa (2001), said, if one can master RNAi,
they could create a way to transfer knowledge, and memory, from one person to another, with
enough research. The evidence shows how, RNAi can be directly linked to memory; because it
can modify the cell, to have certain properties, which means that memory can be created.
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Learning.
The fruit fly, Drosophila melanogaster, demonstrates its associative learning abilities in
both classical and operant conditioning paradigms. Efforts to identify the neural pathways and
cellular mechanisms of learning generally focus largely on olfactory classical conditioning.
Results derived from various genetic and molecular manipulations provide considerable evidence
that this form of associative learning depends critically on neural activity and cAMP signaling in
brain neuropil structures called mushroom bodies (Siwicki & Ladewski, 2003).
Operant conditioning, under experimental studies, reveals a controversial relationship
between associative learning and possible motor learning. Motor learning and its underlying
neural substrates, although extensively studied in mammals, is still not explicit in invertebrates.
The visual discriminative avoidance paradigm of Drosophila at the flight simulator has been
widely used to study the flies' visual associative learning and related functions, but it does not
explain the motor learning process (Wang, Li, Feng, & Guo, 2003). In addition, the visual and
olfactory cues used during navigation by livestock species do not offer a clear explanation.
Evidence suggests that pigs do not acquire and maintain the use of visual cues while foraging.
Other studies suggest that swine can learn multiple-choice spatial tasks, but that the first problem
encountered influences their ability to learn subsequent tasks (Croney, Adams, Washington, &
Stricklin, 2003). In non-associative learning, an animal learns about the properties of a stimulus
presented alone. It either decreases (habituation) or increases (sensitization) its responses to that
stimulus after repeated exposure (Siwicki & Ladewski, 2003).
Many believe hierarchical organization is a distinctive feature in brain information
processing, and the underlying mechanism of the formation of multi-layered neural circuits is an
essential problem in brain science. The established theoretical method of training multi-layered
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neural networks is the well-known back propagation algorithm. Although this method is very
powerful, and has succeeded in many application studies, several problems make it biologically
implausible. Error signals in the post-synaptic neuron propagate backwards to the pre-synaptic
neuron against the spike flow. Watanbe, Masuda and, Aihara (2003) introduce a biologically
plausible method of implementing reinforcement learning to multi-layer neural networks. The
key idea is to localize spatially the synaptic modulation induced by reinforcement signals,
proceeding downstream from the initial layer to the final layer (Watanabe, Masuda, & Aihara,
2003). See Figure 1 in the appendix.
Experimental Design
The independent variable in the experiment was generational cannibalism, which means
the generation of the tested planaria. The levels of the independent variable were first through
fifth generation planaria, each feeding the next generation. The control group was the first
generation. For every level of the independent variable, there were ten trials. The dependent
variable was learning, as measured by the response to stimuli, which was measured in units of
time. The amount of food given to the planaria, the amount of water they were kept in, and the
number of planaria they were kept in contact with remained constant.
Materials and Methods
Technology Component
Apparatus.
The apparatus in this experiment was a pulsing light. A pulsing light blinks on and off at
a specified frequency (a given number of on and off switches per second, measured in hertz.)
The light in this circuit is designed to be very intense, and to blink on and off repeatedly. The
shorter bursts of light energy stimulate the test subject differently than a continuous beam. This
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design also makes it easier to measure exactly the amount of time in between bursts, because the
apparatus can be precisely designed, as opposed to switching a light on and off. This makes the
experiment much more precise, as the exact amount of light applied (time of exposure) is easily
determined. This circuit operates by means of a feedback loop of three logical “not” gates. This
loop feeds a MOSFET (an electronic component which allows current to flow in certain
situations), which either turns on or off current powering a light. The times when the MOSFET
are on can be determined by changing the values of the capacitor and two resistors in the
feedback loop. The apparatus allows for total control, limited only by available resistors and
capacitors. See Figure 2 in the appendix.
Operation.
To operate the circuit, the desired frequency for light pulsing must be decided. In
addition, the required resistors and capacitor must be obtained as determined by the formula F =
.556 / RC. These required resistors and capacitor must then be placed into the circuit, thus
replacing current ones. Then, the circuit must be placed with the light facing the desired test
subject, and the wires for the battery must be placed in the labeled buses to activate the circuit.
See Figure 3 in the appendix.
Procedures and Materials
The materials needed for this experiment were 50 brown planaria (dugesia tigrina), 6
Petri dishes, and 10 bottles of spring water, latex gloves, 1 assembled circuit, and 1 small stone.
Prior research determined the levels. Any change seemed to cap off approximately five
generations. Planaria fed to the next generation was approximately constant; this determined the
number of planaria tested on for the prior level. In addition, wearing gloves throughout the entire
experiment insured safety.
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Ten planaria were taken out of their container and placed into a Petri dish, and labeled
generation one. This was repeated four more times for every generation, increasing the
generation number by one each time.
One planaria from the Petri dish marked “generation one” was placed onto a separate
Petri dish and was then stimulated by the circuit. The time that it took to evade the bright light
emitted by the circuit was then recorded. This was repeated nine times for a total of ten repeated
trials. Ten planaria were then put into a bowl and ground up with a rock. The generation marked
“two” fed on the resulting mixture. This was repeated for four additional levels of the
independent variable, replacing the generation number in the above paragraph with one more
each time, and omitting crushing the generation five planaria, as there was no generation “six”.
Results
Data
The Effect of Generational Cannibalism on the Learning Rate of Planaria
Generation
(Generation
#)
Time to respond to stimuli (seconds)
Average
1
2
3
4
5
6
7
8
9
10
time to
(tri
responds
als
to stimuli
)
(seconds)
I
24 29 23
24
31
22
19
28
46 24
27
II
16 23 19
16
26
28
27
23
23 18
21.9
III
18 16 14
16
21
24
21
26
14 22
19.2
IV
15 22 22
16
15
14
18
18
16 17
17.3
V
16 19 14
14
15
16
17
14
15 12
15.2
As the generation number went up, the time to respond to stimulus went down
significantly. The planaria seemed to make more mistakes at lower generations. They would go
in one direction and turn around, but near the higher generations, they would go in one direction,
at a fast pace, without doubling back.
Statistics
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See the statistics table in the appendix (Table 1). The effects of generational cannibalism
on the learning rate of planaria are summarized in the table above. As the generation number
became greater, the planaria took less and less time to respond fully to the stimuli. The change
was noticeable from the first generation to the second, but after that, it began to decrease slowly
with each generation. The final generation’s mean was nearly half of the first’s (27 compared to
15.2). The variance of the results also went down with each generation as well. At first, there
was a large variance (7.56), which then went gradually decreased, ending up at 1.93 in
“generation five”. The Statistical Package for the Social Sciences (Windows version 11.0.1) was
used to perform the ANOVA test in order to test the following null hypothesis at the .05 level of
significance: The learning rate of planaria is not significantly affected by generational
cannibalism. The null hypothesis was rejected (p = 0.0011 < α = .05 at df = 3). The data did
support the research hypothesis that generational cannibalism in planaria will cause a significant
change in learning.
Graphs
See Figure 4 in the appendix.
Discussion
Analysis
As the number of generations increased, the time it took the planaria to responds to
stimuli decreased. The independent variable was generational cannibalism, planaria often ingest
each other in the wild, and when they ingest other planaria, they acquire RNAi, which can
facilitate the transfer of learning. The topic and dependent variable was transfer of learning in
planaria. Planaria have been used in many experiments to see how they respond to stimuli,
because they can be easily conditioned. They easily learn from stimuli and have a rudimentary
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memory system. This learning is transferred through RNAi, which is provided by generational
cannibalism. RNAi occurs when double-stranded RNA that is expressed within cells is cleaved
into smaller protein-bound fragments that hybridize to endogenous cellular sequences, resulting
in selective degradation of specific endogenous mRNAs and the consequent suppression of
individual gene functions (Smalheiser, Manev, & Costa, 2001). What RNAi essentially does, is
to turn off and on certain genes, such as in this case, when the gene for the knowledge that the
planaria in the prior generation held was turned on. What this means is that planaria can learn by
ingesting other planaria who have learned something in the past. In “generation one”, the value
46 was a significant outlier (z = 2.51, critical value of z = 2.29, p < .05) and thus was removed
from calculations. This point may have occurred because a planarian was damaged, or because it
had a different reaction to the stimulus than the other planaria.
The findings of this experiment were similar to those identified by prior research
(McConnell, 1966). The experiment proved that planaria respond to and learn from stimuli, and
that this knowledge can then be transferred through RNAi to other planaria. There are several
implications related to humans for this experiment. If RNAi could be harnessed, humans would
be able to turn off genes that are considered bad, and turn on genes that are considered good.
This could lead to curing many diseases. The knowledge gained from experimenting with natural
instances of RNAi is a helpful step in the right direction, towards being able to use and apply the
findings.
Summary
The purpose of the experiment was to determine whether intelligence would be
transferred through cannibalism in planaria. The major finding of the experiment was that
planaria do gain intelligence from ingesting other planaria. The hypothesis was supported by that
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data, and the null hypothesis was rejected. This experiment correlates well with what was found
out in prior experiments, that planaria learn through cannibalism by means of RNAi. The
possible explanation for these findings is that planaria do learn from other planaria, and that they
do this through RNAi switching on and off certain genes in their cells. There are several possible
experiments that could be done with the planaria besides the one performed, to measure transfer
of intelligence. One possible future experiment would be to observe behavior over ten
generations instead of five, in order to see whether and when the learning hits a limit. In addition,
one could attempt to use different stimuli to see if the same results are accomplished.
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References
Best, J. S. (1960, August). Diurnal cycles and cannibalism in planaria. Science, 131,
1884-1885.
Brown, H., Dustman, R., & Beck, E. (1965, December). Sensitization in planaria.
Physiology & Behavior, 1(3-4), 305-308.
Brown, H., Dustman, R., & Beck, E. (1965, December). Experimental procedures that
modify light response frequency of regenerated planaria. Physiology & Behavior, 1(3-4),
245-249.
Croney, C., Adams, K., Washington, C., & Stricklin, W. (2003, October). A note on
visual, olfactory and spatial cue use in foraging behavior of pigs: indirectly assessing
cognitive abilities. Applied Animal Behavior Science, 83(4), 303-308.
McConnell, J. (1966). Comparative physiology: Learning in invertebrates. Annual
Review of Physiology, 28, 107–136.
Sanjuan, M., Alonso, G., & Nelson, J. (2003, November). Blocked and test-stimulus
exposure effects in perceptual learning re-examined. Behavioural Processes, Article in
press, corrected proof.
Smalheiser, N., Manev, H., & Costa, E. (2001, April). RNAi and brain function: Was
McConnell on the right track? Trends in Neurosciences, 24(4), 216-218.
Siwicki, K., & Ladewski, L. (2003, September). Associative learning and memory in
Drosophila: Beyond olfactory conditioning. Behavioural Processes, 64(2), 225-238.
Wang, S., Li Y., Feng, C., & Guo, A. (2003, August). Dissociation of visual
associative and motor learning in Drosophila at the flight simulator.
Behavioural Processes, 64(1), 57-70.
Watanabe, M., Masuda, T., & Aihara, K. (2003, September). Forward propagating
reinforcement learning: Biologically plausible learning method for multi-layer networks.
Biosystems, 71(1-2), 213-220.
15
Appendix
Figure 1
16
Figure 2
Figure 3
17
Figure 4
The Effect of Generational Cannibalism on Learning in Planaria
Time to respond to stimuli
(seconds)
30
25
20
15
10
5
0
1
2
3
4
5
Generation (Generation Num ber)
As the generation of planaria increases, the time to respond to stimulus decreases.
Table 1
The Effect of Generational Cannibalism on the Learning Rate of Planaria
Descriptive
Information
Mean
(Amount of
time to
adjust to
stimulus in
seconds)
Standard
Deviation
Repeated
Trials
1
27
2
21.9
Generation Number
3
19.2
7.56
4.43
4.21
2.79
1.93
10
10
10
10
10
Results of ANOVA test
df = 3, F = 5.51,
p = 0.0011 < α = .05
4
17.3
5
15.2