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The Effect of Induced Cannibalism on Learning in Planaria
Victor Mircea
TJHSST
Biology I, Period 6
Dr. Wood
March 27, 2004
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
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) could be reliably conditioned
to 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 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 double-stranded 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.
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 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 postsynaptic 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.
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
three-branched 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 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.
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 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 desired for the pulsing of the light
must be decided. And, what resistors and capacitor are required (as found by the formula
F = .556 / RC) must be obtained. The required resistors and capacitors must be placed
into the circuit in place of the 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), six petri dishes, ten bottles of spring water, latex gloves, one assembled circuit,
one small stone.
The levels were determined based on prior research. In prior research any change
seemed to cap off approximately five generations. The amount of planaria that was fed to
the next generation was approximately constant, and was determined by the number of
planaria that were tested on for the prior level. Also, safety was kept by wearing gloves
throughout the entire experiment.
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. After they were ground up, they were fed to the generation marked two. 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 6.
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 many 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 and not double back.
Statistics
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 slowly decrease 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), but then it
went down finally ending up at 1.93 in generation 5. The ANOVA test was used 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 memory system. And, this learning is transferred through
RNAi, which is provided by generational cannibalism. RNAi occurs when doublestranded 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 (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 I, 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 the experiments done by prior
researchers on similar subjects. The experiment proved that planaria respond to and learn
from stimuli, and that this knowledge can then be transferred through RNAi to other
planaria, which matches what the literature claims (McConnell, 1966). 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,
because the more is known about a subject, the easier it can be used and applied.
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 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
experiment would be to repeat this experiment, and to go out for ten generations instead
of five, and to see whether and when the learning hits a limit. Also, one could attempt to
use different stimuli to see if the same results are accomplished.
Literature Cited
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), 225238.
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.
Appendix
Figure 1
Figure 2
Figure 3
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 Number)
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
4
17.3
5
15.2
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