Download ASSOCIATIVE LEARNING IN ANTS: ODOR LEARNING VS. COLOR

ASSOCIATIVE LEARNING IN ANTS:
ODOR LEARNING VS. COLOR LEARNING IN NOVOMESSOR COCKERELLI
By
Sky Harris Sobol
____________________
A Thesis Submitted to The Honors College
In Partial Fulfillment of the Bachelor’s degree
With Honors in
Neuroscience and Cognitive Science
THE UNIVERSITY OF ARIZONA
December 2015
Approved By:
___________________
Dr. Wulfila Gronenberg
Department of Neuroscience and Cognitive Science
Abstract
Associative learning is a form of learning where an animal learns to associate a
stimulus with a behavioral response. Associative learning has been generated in the
laboratory for many species, including insects, by using classical conditioning.
Previous experiments show that ants can learn to associate odors and colors with a
sugar reward. When ants are trained to associate a stimulus with a sugar reward,
they exhibit the proboscis extension response (PER) to the stimulus alone, but it
was unknown whether ants are better at color association or odor association. In
order to test this, two separate experiments were undertaken. The first used
classical conditioning to train ants to associate a sucrose solution with an odor. The
second used classical conditioning to train two groups of ants. The first group was
trained to associate a sucrose solution with a blue light. The second group was
trained to associate a sucrose solution with a green light. For both experiments a
significant percentage of ants demonstrated learning by exhibiting PER when
presented solely with the odor or light. There was, however, no significant
difference between the percentages of ants that demonstrated learning from the
blue light, the green light, or the odor.
Introduction
The American Psychological Association defined learning as “the acquisition of
skill or knowledge”, and memory as “the expression of what was acquired” (Encyclopedia of Psychology, 2000). Learning and memory are critically important to many
species, including humans. Indeed, human life and society would be unrecognizable as
we know it if we were unable to retain and use new information. Accordingly, many
studies have focused on human learning and memory because it is so critical to our daily
lives.
One relatively simple and well-understood form of learning that has been
observed in many species, including humans, is associative learning.
Associative
learning is when a behavioral response is taught to be associated with a stimulus.
Associative learning is a simple form of learning that can be easily generated (hence,
researched) in a laboratory setting. Classical conditioning is one of several techniques or
procedures that can be used to establish associative learning. It is a common, wellestablished technique used in many psychological experiments. The best-known example
of an experiment in classical conditioning is the famous experiment involving Pavlov’s
dogs (Pavlov, I.P., 1928). Every time Pavlov’s dogs were presented with food (the
unconditioned stimuli), the dogs would drool. When Pavlov rang a bell absent the
presentation of food, the dogs did not drool (the conditioned stimuli). The conditioning
period took place when the unconditioned stimuli and the conditioned stimuli were
paired, that is, Pavlov would ring the bell every time the dogs were presented with food.
After multiple sessions in which the two stimuli were paired, the dogs would respond to
the ringing of the bell by drooling even when there was no food offered. In other words,
the previously conditioned stimulus could now cause the response on its own. The dogs
(or certain parts of their brains) had associated the sound with a reward.
Classical conditioning experiments have been performed on many animals,
including vertebrates and invertebrates, and many conditioning experiments have been
performed on insects. Insects are good subjects for studying simple learning processes at
the behavioral level. They have the advantage of not costing much to acquire, they are
easy to maintain in a laboratory, and there is less of an ethical concern when working
with them. In addition, insects’ brains do not have the complexity of most vertebrates’
brains, thus making it easier to find out where in the brain the learning associations occur.
One of the first well-documented classical conditioning experiments was performed on honeybees (Takeda, 1961). In this experiment (as explained by Masumoto et
al. 2012), bees were conditioned to associate sugar water with an odor. Whenever the
antenna or the mouthparts of bees come in contact with sucrose solution, bees reflexively
extend their proboscides (tubular mouth parts) to the sucrose solution. This is known as
the proboscis extension response (PER), and it has proven to be an excellent indicator of
conditioned responses in classical conditioning experiments because untrained bees do
not extend their proboscis to an odor alone. By pairing sucrose solution presentation with
an odor, Takeda was able to condition the subject bees to exhibit the PER when presented
with only the odor.
Experiments have established that ants also respond to classical conditioning. In
one experiment ants were conditioned to respond to an odor (Guerrieri and d’Ettorre,
2010). When the ants were stimulated by the presentation of a sucrose solution, they
extended their mouthparts (maxilla-labium; the homologue of the bees’ proboscis) to
obtain the sucrose solution. The researchers then performed six conditioning trials pairing
the unconditioned stimuli (the sucrose) with the conditioned stimuli (an odor). After
completion of the trials, the ants were far more likely to extend their maxilla-labium
when presented with the conditioned odor than they were when presented with a completely unfamiliar odor.
Although ants seem to rely more on their sense of smell than vision, ants do seem
to have color vision, and were able to respond to wavelengths of light from 360 nm to
620 nm (Marak and Wollen, 1965). Aksoy and Camlitepe (2012) also found that ants can
respond to multiple wavelengths in the visual spectrum. This shows that ants are able to
perceive most of the colors on the visible spectrum, including green and blue lights.
However, it is still unknown if they can actually discriminate blue and green light (a
question beyond the scope of the current thesis). Visual learning has also been tested in
ants using classical conditioning with visual stimuli instead of odors. One experiment
found that Formica unicularia could associate the color green with a reward using
absolute conditioning, which is classical conditioning as defined above. They also found
that the ants associated the color green with a reward in differential conditioning.
Differential conditioning is similar to absolute conditioning, except the animals are
trained to respond to one stimulus while ignoring another similar stimulus, such as
learning green light, while ignoring a blue light (Camlitepe and Aksoy, 2010).
These experiments have shown that ants are capable of being classically conditioned to both color and odor stimuli.
The experiments I conducted investigated
whether ants are better and more easily conditioned to color or to odor. My hypothesis is
that ants will be better at odor learning than they are at visual learning. This is because
most ants are small terrestrial species that rely on many different food sources. They rely
mostly on scent to gather their food, especially since leaf litter may often obstruct their
vision. In that instance, sight might not be used at all. For these reasons, scent is more
likely an important sense than sight. Accordingly, ants would be able to learn more
quickly through scent-based associations.
Materials and Methods
Animals
The ant species used for this experiment was Novomessor cockerelli, a
relatively large ant that is common in and around Tucson. This species is active
during the cooler parts of the day and, in Tucson, AZ, is active throughout the year.
Ant workers were collected from three different colonies were used for this
experiment (Colony A, B and C). All three colonies were located along a stretch of
First Avenue North of River Road in Tucson, 85718. 20-40 ants were used from
each colony.
The ants were housed in plastic boxes coated with Fluon (Asahi Glass
Fluoropolymers) to prevent the ants from climbing on the container wall. The ants
were provided with water at all times, and sugar water when they were not being
starved.
Starvation and Harnessing
Before the conditioning experiment began, sugar water was removed from
the ants’ containers for three days. After three days, 14 ants were removed from the
containers with soft forceps and placed in a vial. After the fourteen ants were
removed, the sugar water was put back in the container. The vial was then placed in
ice for ten minutes in order to anesthetize the ants. After ten minutes, the ants were
harnessed. The harnesses were made out of plastic pipette tips and strips of duct
tape (Figure 1). The ants were harnessed at the thorax, so they could move their
heads and front legs, but not the rest of their bodies.
After harnessing, the ants were placed in a container with a moist paper
towel, so they would not dry out. They were left in the container for two days before
the odor experiments.
Fig 1. Harnessed ants in preparation for conditioning
Odor Conditioning
The odor used was a basic perfume compound called Fruity (Spinnrad AG,
Germany). Filter paper was soaked with five l of odorant and placed in a 10ml
plastic syringe without a plunger and attached to an aquarium pump in order to
pump the odorant toward the ants.
Before testing, the ants were preliminarily tested to see if they showed
interest in a 30% sucrose solution offered on a toothpick. Interest was measured by
whether the ant exhibited the proboscis extension response (PER), which is the
extension of the “tongue” in order to lick the sugar water. The ants that did not show
interest were placed back in the container and not used for the experiment. The ants
that did show interest were placed in a cardboard carousel (Figure 2). The carousel
was comprised of 10 sectors, each one holding one ant and connected to a vacuum
attachment so the odor did not drift into other compartments or linger near the
ants.
After all of the ants were attached to their compartments with wax, the
conditioning began. Each trial comprised the following sequence: the conditioned
stimulus (the odor) was presented to the ants through the aquarium pump for seven
seconds. Five seconds after the onset of the odor stimulus, the unconditioned
stimulus (the sugar water) was presented. Whether the ant demonstrated the PER
during the conditioned stimulus or during the unconditioned stimulus was
recorded. If an ant responded with the PER during the first 5 seconds of odor
presentation, before the sugar water stimulus, this was counted as a conditioned
response.
One experiment consisted of ten trials in total. Between each trial, there was
a five-minute wait before the next trial began. If an ant did not exhibit the PER for
the unconditioned stimulus for three trials, it was assumed to be satiated or
otherwise unmotivated and was excluded from the data.
After the odor conditioning was finished, the ants were kept in their
harnesses and placed back in the harness container. The next day they were tested
for their memory by exposing them to the odor stimulus for five seconds. A record
was made noting whether or not they exhibited the PER.
Fig. 2. Cardboard carousel and Aquarium pump for odor conditioning.
Visual Conditioning
Ants were harnessed as above and then placed in a visual carousel (Figure
3) and secured using paraffin wax. The carousel contained 8 cup-shaped compartments, one for each ant. A light source with blue and green LED lights could be
placed on top of the ants’ compartments so that diffuse light would shine into the
ants’ compartments.
Unlike the odor conditioning, each ant was rated for activity before the
conditioning. Ants were rated on a scale from 1 to 5, with 1 being the least active
(barely any movement) and 5 being the most active (exaggerated movement of
antenna, head and legs).
After all of the ants were attached to their compartments with wax, the
conditioning began. Each ant was assigned a color, either blue or green, as the
conditioning stimulus. The conditioning stimulus (the assigned light) was presented
to the ant for seven seconds. Five seconds after the onset of the respective light
stimulus, the unconditioned stimulus (30% sugar water) was presented. Whether
the ant exhibited the PER during the conditioned stimulus and/or during the
unconditioned stimulus was recorded.
There were ten trials in total, with inter-trial intervals of five minutes. After
ten trials, each ant was presented with the color stimulus that they were not tested
on. If an ant did not exhibit the PER for the sugar stimulus for three trials, it was
excluded from the data.
After the color conditioning was completed, the ants were kept in their harnesses
and placed back in the harness container. The next day they were tested for their
memory. They received their conditioned visual stimulus for five seconds, and then,
as a control for stimulus generalization, the respective other light stimulus for five
seconds (the color they were not trained to associate). It was then recorded whether
or not they exhibited the PER.
Data Analysis
Odor Training Analysis
Using Excel, each ant’s response to each trial was recorded by placing a 1 in
the excel sheet to indicate whether the ant showed the PER for the unconditioned
stimulus, and a 0 if the ant did not show the PER for the unconditioned stimulus. For
each trial, the number of responses was summed and then divided by the total
number of ants for that trial in order to get an average for that trial. This was done
for all ants for all ten trials.
A graph was then constructed from the average percent learned vs. the trial
number. A logistic regression was run, and the equation and R2 value were
calculated for the data. A p-value was taken from the logistic regression in order to
determine whether the ants were learning or whether they were just responding
randomly. Standard deviation for each trial was then calculated using Excel.
Fig. 3. Carousel for visual conditioning.
The ants that responded any time on any of the last three trials were used for
the memory test. For the memory test, the number of ants that showed the PER was
added up and then divided against the total number of ants in the memory test,
giving a percentage of ants that remembered their conditioning. The memory test
percentage was placed on the graph in it’s own column.
Visual Training Analysis
The data analysis for the visual training was very similar to that used for the
odor training. There were, however, two sets of data that needed to be analyzed: the
ants trained on the blue light, and the ants trained on the green light.
In similar fashion to the data analysis for the odor experiment, and for each
set of data, using Excel, each ant’s response to each trial was recorded by placing a 1
to indicate whether the ant showed the PER for the unconditioned stimulus, and a 0
to indicate whether the ant did not show PER for the unconditioned stimulus. For
each trial, the number of responses was summed and then divided by the total
number of ants for that trial in order to get an average for that trial. This was done
for all the ants for all ten trials.
For each color, a graph was then constructed from the average percent
learned vs. the trial number. A logistic regression was run, and the equation and r2
value were calculated for the data. A p-value was taken from the logistic regression
in order to determine whether the ants were learning or whether they were just
responding randomly. Standard deviation for each trial in each color was then
calculated using Excel. In order to determine whether the ants learned green light
differently from the blue light data, a Mann Whitney U test was conducted. The null
hypothesis was that the two sets of data would not be different. The raw number out
of ten for each ant was used as data points for the Mann-Whitney U test. If the two
sets of data were not different from one another and the distribution was normal,
the Student’s t-test was conducted in order to further determine whether the ants
learned the colors in the same way. A further Student’s t-test was conducted for the
total percentage for last three trials.
If an ant responded to the color on any of the last three trials, it was then
tested on the opposite color to see if the ant could discriminate between different
colors. The number of responses to the alternate color were summed and divided by
the total number of ants tested in order to determine the percent of ants which
indiscriminately responded to each color.
A memory test was conducted for the ants trained on the green light, and the
ants trained on the blue light. The ants that responded any time on any of the last
three trials were used for the memory test. For the memory test, the number of ants
that exhibited the PER was added up and then divided by the total number of ants in
the memory test, producing the percentage of ants that remembered their
conditioning after 1 day. The memory test percentage was placed on the graph in it’s
own column.
Odor and Color Data Comparison
Because it was found that the ants did not learn green light and blue light
differently, the green light and blue light data were combined into one data set,
titled “Color Training.” Both the odor and color data were placed on the same graph,
along with their respective memory test data.
In order to determine whether the ants learned the colored lights the same
way, a Mann-Whitney U test was conducted in order to compare the two sets of
data. Because the data was found to be normal, a Student’s t-test was conducted in
order to further analyze whether the data sets were different. Each individual ant’s
data for the ten trials was used as data points for the Mann-Whitney U test and the
first Student’s t-test.
A Student’s t-test was also conducted on the last three
percentage points
Results
Results for Odor Conditioning
Forty-eight ants were used for this experiment (Figure 4). No ants
responded the first odor presentation. Less than 5% of ants responded to the odor
for Trial two and three. There was a 10% increase in responses for Trials four to six.
In Trial 7, 24% of ants responded exhibited the response to the odor. In Trial 8, 22%
of the ants exhibited the response to the odor. In Trial 9, 22% of the ants exhibited
the response to the odor. In Trial 10, 24% of the ants exhibited the response to the
odor.
A linear regression (y=0.03x-0.02; R2 = 0.85) of the data shows that learning
takes place after the first training cycle. A logarithmic equation (R2 = 0.7362; Chisquared value: 16.12; degrees of freedom: 1, p = <.0001) may be a better fit and it
would suggest that the learning performance tapers off over time and may approach
a maximum amount 30%. There was a large standard deviation for each individual
trial.
Ants that responded to the odor on Trials 8, 9, and/or 10 were selected and
used for the memory test. 13 out of 48 ants were selected for the memory test. 30%
of ants showed PER on the memory test out of the ants selected for the memory test.
When compared with the total number of ants trained, 8% of ants responded to the
memory test.
Fig. 4. Proportion Learned vs. Trial Number for ants trained on odor. The ants showed nonrandom learning (y=0276x-.0172, R2 = .85402). Ants that responded to the odor on Trials 8, 9,
and/or 10 were selected and used for the memory test. 13 out of 48 ants were selected for the
memory test. 0.3 was the proportion of ants that showed PER on the memory test out of the ants
selected for the memory test. When compared with the total number of ants trained, 0.083 was
proportion of ants that responded to the memory test.
Results for Visual Testing
For this experiment (figure 5), 27 ants were trained to associate a green
light with a sugar reward and 26 ants were trained on a blue lights. There was no
learning for the green on the first five trials. In trial six, 4% of the ants tested on
green exhibited the response to the light. Learning increased after the sixth trial. In
Trial 7 and 8, for the ants tested on the green light, 16% exhibited the response to
the light. In trial 9, for the ants tested on green light, 18% of the ants exhibited the
response to the light. In Trial 10, for the ants tested on green light, 29% of the ants
exhibited the response to the light.
No ants showed a response to the blue light in Trial 1. In Trial 2 and 3, 4% of
the ants test on blue exhibited the response to the light. In Trials 4-6, for the ants
tested on the blue light, 7% of the ants exhibited the response to the light. A large
increase in learning occurred after Trial 7 for the ants trained on blue. In Trial 8, for
the ants tested on blue light, 30% of the ants exhibited the response to the light. In
Trial 9, for the ants tested on blue light, 29% of the ants exhibited the response to
the light. In Trial 10, for the ants tested on the blue light, 29% of the ants exhibited
the response to the light.
It was found that learning occurred for the ants trained on both colors
(green: chi-square value 25.73, degrees of freedom: 1, p-value < .0001; blue: chisquare: 25.49, degrees of freedom:1, p < .0001) The R2 value was 0.8256 (green)
and 0.7362 (blue), respectively, which shows the data for both colors fit a
logarithmic equation.
The data distribution was normal and there was no significant difference
between how the ants learned green and blue (Z-score: .8451, p-value: .39532, Uvalue was 303). The t-test showed that there was no significant difference between
how ants learned green and blue stimuli (t-value: 1.025997, P-value: 0.154868).
The data for trials 8 and 9 seemed to suggest that at these time points blue
might be learned faster than green; however, a t-test conducted for the last three
percentage points for Trials 8, 9, and 10 did not reveal any significant difference in
the way the ants learned blue vs green (T-value: 2.054987, P-value was .05436).
Seventy-seven percent of the ants trained on the green light and 83% of the ants
trained on the blue light responded to the respective alternate color (Figure 5).
Fig. 5. Proportion Learned vs. Trial Number for ants trained on blue and trained on green. Green
diamonds represent ants trained on green, and the blue squares represent ants trained on blue.
Both the blue-trained ants and green-trained ants showed learning (Blue: y= 0.0367x-.0677, R2 =
0.86525; Green: y= 0.0315x- 0.09, R2 = 0.81291). There was no significant differences between two
the two sets of data (t-value: 1.025997, P-value: 0.154868) Ants that responded to the colors for
both sets of data on Trials 8, 9, and/or 10 were used for the alternate color test (percentage
responded to other color) and the memory test. 13 out of the 26 ants trained on green were
selected for the memory test. The proportion of ants that showed PER for the memory test was
0.23. When compared with the total number of ants trained on green, 0.115 was the proportion of
ants that responded to the memory test. 14 out of the 27 ants trained blue were selected for the
memory test. The proportion of ants that showed PER was 0.50. When compared with the total
number of ants trained on blue 0.259 responded to the memory test
Ants that responded to the colors for both sets of data on Trials 8, 9, and/or
10 were used for the memory test. 13 out of the 26 ants trained on green were
selected for the memory test. 23% of the ants showed PER for the memory test.
When compared with the total number of ants trained on green, 12% of the ants
responded to the memory test. 14 out of the 27 ants trained for blue were selected
for the memory test. 50% showed PER. When compared with the total number of
ants trained on blue 26% responded to the memory test.
Fig. 6. Proportion learned vs. Trial Number for ants trained on odor and trained on color (The
combined data of blue and green). Yellow diamonds represent the ants trained on odor and the
red squares represent the ants trained on color. There was no significant difference between the
two data sets (t = 0.40353; p = 0.34714.). Only the ants that responded at some point on the last
three trials were selected for the memory tests. 13 out of 48 ants trained on odor were selected
for the memory test. 0.3 was the proportion of ants that showed PER on the memory test out of
the ants selected for the memory test. When compared with the total number of ants trained,
0.083 was proportion of ants that responded to the memory test. The memory test data for the
ants trained on green and blue was combined. 27 of the 53 ants trained on color were selected for
the memory test. 0.37 was the proportion of the ants that showed PER on the memory test out of
the ants selected for the memory. When compared with the total number of ants trained, 0.18 was
the proportion of ants that responded to the memory test.
Comparison of Odor and Color
As shown in figure 5, no difference in learning performance was found for the
two colors. All the ants trained for either green or blue were therefore combined
and together compared to the learning performance of the ants trained to associate
an odor (figure 4). Figure 6 shows that the data distribution was normal and that
there was no significant difference between how the ants learned odor vs. colors (Zscore: -.374, p-value: 71138, U-value: 1216.5). In order to further test whether the
ants learned odor differently from the manner in which they learned color, a t-test
was conducted. There was no significant difference between the data (t = 0.40353;
p = 0.34714.
A t-test was conducted for the last three percentage points for trials 8, 9, and
10 in order to test whether the data was different. There was no significant
difference between the data (t= 1.131023, p= 0.16031)
The memory test also did not suggest any significant differences: of the ants
selected for the memory test, 30% of the odor-trained ants and 37% of the colortrained ants remembered their respective association one day after the training.
Discussion
Odor Conditioning
In the current study, 24% of the ants exhibited the proboscis extension
response (PER) to the associated odor after ten trials. The high R2 and the low pvalue calculated from the logistic expression indicate that the pattern of responses
to the odor stimuli was not random, and that the ants exhibited a pattern of
responses that likely signify learning. In a study on the ant species Camponotus
aethiopus that was conducted in a similar manner as the current study, researchers
found that, “When a conditioned stimulus was… paired with the unconditioned
stimulus that… [PER] increased significantly with successive trials” (Guerrieri and
d’Ettorre 2010). That pattern is similar pattern to the one followed by the ants in
the current study, and supports the finding that ants are capable of associative
learning.
Even though the patterns of learning were similar between those in the
current study and those in the Guerrieri and d’Ettorre study, the Guerrieri and
d’Ettorre study resulted in a much higher rate of learning and a quicker onset of
learning compared to the current study. In Trial 2 of the Guerrieri and d’Ettorre
study, 20% of the ants exhibited the PER, and by the sixth and final trial,
approximately 45% of the ants exhibited the PER. One factor that might have led to
a higher success rate in the Guerrieri and d’Ettorre study was that the researchers
used an inter-trial interval of ten minutes. In the current study, the inter-trial
interval was only five minutes. In a study on honeybees, Menzel (1999) found that a
ten-minute inter-trial interval was ideal for consolidation of memory. While a
review of the literature concerning ants revealed no ideal inter-trial interval for, a
ten-minute inter-trial interval may have offered the ants in the instant study a more
effective opportunity to consolidate memory.
The ants in the current study were also a different species from the ants used
in the Guerrieri and d’Ettorrre study. This, too, may account for the difference in
results.
In addition, the current study had a larger standard deviation for each trial
than that of the Guerrieri and d’Ettorre study. Guerrieri and d’Ettorre used ants
from the same colony in each of their experiments, whereas I used ants taken from
different colonies as the study progressed. I observed that ants from different
colonies responded differently, producing drastic differences in data between
colonies. (These differences are discussed in further detail, below.) The use of ants
from more than one colony likely caused a larger standard deviation.
Although not explicitly stated by Guerrieri and d’Ettorre, their study was
apparently conducted during a shorter time period than my study and with true
colonies. My study took place over ten weeks, and I did not have a full colony, only a
group of workers without queen. My ants did not have brood to care for, and this
might have reduced their motivation for foraging and learning. This factor and the
longer time period used may have caused changes in the ants, thus further
contributing to a larger standard deviation.
Another possible source of differences in results between these studies is
related specifically to the manner in which the ants in my study were handled. In the
beginning phases of the experiment, the ants were inverted (held upside down)
during the conditioning. Although the ants’ initial responses to the odor were
robust, it became apparent that the ants were exhibiting the PER in response to
being inverted. As a result, it was not possible to conclusively determine whether
the ants were responding to the odor, and the data was discarded. A similar
problem may have resulted from the air puff used to dispense the odor not being
controlled for. This may have caused in the ants to exhibit the PER in response to
the air puff rather than the odor. Data obtained with this method was used in the
study, but in future studies it is recommended that the air puff be controlled for by
testing the ants on air puffs alone and on the odor alone in order to make sure the
ants are only responding to the odor and not just the air puff.
When compared to the 45% of the ants in Guerrieri and d’Ettorre study that
exhibited the PER when exposed to the odor one hour after the conditioning trials,
30% of the ants in the current experiment exhibited the PER when exposed to the
odor one day after the conditioning trials. The percentage of ants exhibiting the PER
in my study was likely lower because of the longer delay between the conditioning
and the memory test. The finding that 30% of the ants remembered the odor after
one day supports the conclusion that it is important for ants to remember certain
smells. This may be the case because these ants are scavengers, and remembering
associations between unusual smells and edible substances may increase their
individual foraging success rate.
Visual Conditioning
The data from the green and blue light studies resulted in a high R2 and low
p-value calculated from the logistic expression. This supported the conclusion that
the learning was not random, and that the pattern of responses likely indicated
learning. A Mann-Whitney U and a Student’s t-test indicated that there was no
significant difference between the ants that learned the green light and the ants that
learned the blue light.
77% of the ants that exhibited the PER when conditioned using only the
green light also exhibited the PER when presented with the blue light. 83% of the
ants that exhibited the PER when conditioned using only the blue light also
exhibited the PER when presented with the green light. These high levels of
responses along with the results of the t-test support the conclusion that the ants
were likely just responding to the presence of light, rather than to its color.
Previous studies have shown that in addition to ants’ ability to be trained to
respond to a particular color, ants can also discriminate between certain
wavelengths of colors. In a study conducted by Aksoy and Camlitepe (2009) it was
found that the ant species Formica cunicularia could discriminate between lights in
the wavelengths of 510 nm and 550 nm. The other species of ant used in this
experiment, the desert ant Cataglyphis aenescens, could not discriminate between
lights in those wavelengths. In a follow-up study conducted by Aksoy and Camlitepe,
it was found that Formica cunicularia could also discriminate between light in the
violet and green wavelengths, and in the green and red wavelengths, but could not
discriminate between lights in the other wavelengths tested from to violet to red
(blue was not tested). A recent electrophysiological study suggests that highly visual
bulldog ants (genus Myrmecia) have a trichromatic color vision system (Ogawa et al.
2015), but it is still not clear if this is true for other ant genera. My study was not
suited to test whether Novomessor cockorelli can perceive or discriminate between
certain colors.
There are many possible reasons why the ants in the current study were
likely not able to discriminate between the blue and green lights. Discrimination
between blue and green lights by ants has not been tested. Formica cunicularia were
unable to discriminate between certain colors, though the colors tested did not
include blue and green. It is also possible that while other species of ants might be
able to discriminate between green and blue lights, that Novomessor cockerelli might
not have that ability.
A possible source of error that may have contributed to the ants being unable
to discriminate between the blue and green lights is that in order to perform the
experiment, the ants were starved and then harnessed. The ants in Aksoy and
Camlitepe experiments were not starved and not harnessed. Starving and
harnessing the ants may have reduced their cognitive abilities, or fatigued them to a
point where making that distinction became too cognitively challenging.
Another reason that the ants might have been unable to discriminate
between the blue light and the green light is that the Aksoy and Camlitepe
experiments were set up differently than the trials in the instant study. Aksoy and
Camlitepe conditioned their ants by setting up a y-maze with two compartments.
One maze compartment contained a sugar reward and particular wavelength of
light, and the other maze compartment contained a different color and no reward.
Aksoy and Camlitepe thus used differential conditioning, which trains the ants to
learn the difference between two colors while my approach did not reinforce
differential color learning.
In future experiments differential conditioning should be used in order to
train the ants to a specific color. In differential conditioning, ants would be exposed
to lights of two different colors, but would only be rewarded for one of the two
colors. This approach will help determine whether the ants are responding to the
color of the light or just the light itself. Adding differential conditioning to the study
could be done in the following manner: In each trial, one color of light would be
shown and a sugar reward would be presented, and then another light color would
be shown and no reward would be presented. The order of the first light and the
reward, and the second light without the reward would then be switched for each
trial in order to make sure the ants were not simply learning the sequence of
events. If the ants exhibited a low percentage of the PER to the unpaired color, but
exhibited a high percentage of the PER to the paired color, then this would
demonstrate that the ants can discriminate between colors.
Odor vs. Color Conditioning
The odor data and the light data were compared using a Mann-Whitney U
and a Student’s t-test. Despite the two data sets differing, it was found that there
was no significant difference between the ants that were trained on the odor and the
ants that were trained on the colored lights.
These results do not support the initial hypothesis, i.e. that ants will be better
at learning odor stimuli than a visual stimuli because ants tend to navigate their
world based on smell.
There are several reasons this could be the case. The first might be that this
particular species of ant might rely on smell and sight equally to navigate the world.
These ants are desert dwellers and the hot sand which covers much of their habitat
may provide fewer odor cures that what ants experience in more humid habitats
(e.g. meadows, forests, etc.). Another possibility, as discussed above, is that ants in a
laboratory setting, especially after being starved and harnessed, might have their
senses and cognitive abilities blunted and may no longer be able to learn and
respond to stimuli as they normally would.
One issue encountered during this experiment was that different colonies
behaved differently. For example, the first colony used was fairly passive and would
often die quickly under stress. In contrast, the second colony was very aggressive
and was difficult to train because they responded so belligerently to the toothpick
on which the sucrose solution was presented. These ants were extremely active and
thrashed around a lot. They were also much worse learners than the ants in the first
colony. This raised a question whether some colonies are better at learning due to
either temperament or the age of the colony. As discussed above, using a variety of
colonies was a likely culprit for the large standard deviation. In future studies,
researchers should consider generating statistics on the various colonies used in
order to see if there are any significant differences in learning ability between
different colonies.
Activity level did appear to influence how well the ants learned. The most
and least active ants tended to be the poorest learners. This might be due to the
temperament or health of the individual ants.
Another student is currently
conducting a study similar to this one, and is rating the ants in the study for activity
levels in order to better determine how activity levels affect learning. This is a useful
technique for future studies.
In future studies related to the classical conditioning of ants, it would also be
helpful to control for more variables, work with a larger number of subjects, and
employ more rigorous methodology. In addition, performing brain lesioning on ants
after they have been conditioned to a stimulus would allow researchers to study
which parts of the ant brain are involved in learning, including learning specific to
odor and colors. There is, however, a possible limitation to lesioning experiments.
As noted above, this study resulted in a relatively small percentage of ants
exhibiting the PER after being trained to the odor or the colored lights. The distress
ants may experience after being lesioned may result in an even lower number of
responses. A much larger sample size and stress-related controls may be necessary
to ensure reliable results.
These types of studies lead to a better understanding of the brain, learning,
and insect behavior. Not only do they shed light on the workings of insect nervous
systems (thus providing models for more complex nervous systems), they also offer
an insight into how ants navigate the world.
That knowledge may also offer a practical, environmental, and commercial
benefit. Ants and humans coexist in almost every environment, often leading to ant
infestations of human homes and workplaces and the subsequent application of
toxic pest control chemicals. If we can gain a better understanding of what ants are
sensitive to and how they learn, this information may some day be used to develop
non-toxic, cost-effective methods of pest control.
Acknowledgements
I would to like thank Dr. Gronenberg for being and excellent and patient thesis
mentor, and Rebekah Keating for aiding me with statistics. I would also to thank
Julia Cazier for aiding me with Photoshop for my figures. I also want to thank my
NSCS advisor, Becca Van-Sickler for advising me on how I should schedule my thesis
credit. And lastly, I want to thank my parents, Mendy and Margie Sobol for their
continued support.
Works Cited
Aksoy, V., & Camlitepe, Y. (2012). Behavioural analysis of chromatic and achromatic
vision in the ant Formica cunicularia (Hymenoptera: Formicidae). Vision
Research, 67, 28-36.
Camlitepe, Y., & Aksoy, V. (2010). First evidence of fine colour discrimination ability
in ants (Hymenoptera, Formicidae). The Journal of Experimental Biology,
213(1), 72-77.
Guerrieri, F. J., & d’Ettorre, P. (2010). Associative learning in ants: Conditioning of
The maxilla-labium extension response in Camponotus aethiops. Journal of
Insect Physiology, 56(1), 88-92.
Kazdin, A. E. (2000). ENCYCLOPEDIA 0F PSYCHOLOGY.
Marak, G. E., & Wolken, J. J. (1965). An action spectrum for the fire ant Solenopsis
saevissima. Nature, 205, 1328–1329.
Matsumoto, Y., Menzel, R., Sandoz, J. C., & Giurfa, M. (2012). Revisiting olfactory
classical conditioning of the proboscis extension response in honey bees: a step
toward standardized procedures. Journal of Neuroscience Methods, 211(1), 159167.
Menzel, R. (2001). Searching for the memory trace in a mini-brain, the honeybee.
Learning & Memory, 8(2), 53-62.
Ogawa Y, Falkowski M, Narendra A, Zeil J, Hemmi JM (2015) Three spectrally distinct
photoreceptors in diurnal and nocturnal Australian ants. Proc. R. Soc. B 282: 20150673
Pavlov, I. P., & Gantt, W. (1928). Lectures on conditioned reflexes: Twenty-five years of
objective study of the higher nervous activity (behaviour) of animals.
Takeda, K. (1961). Classical conditioned response in the honey bee. Journal of Insect
Physiology, 6(3), 168-179.