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How do brain chemicals influence who wins a fight?
Featured scientists: Andrew Bubak and John Swallow from the University of Colorado at
Denver, and Kenneth Renner from the University of South Dakota
Research Background:
In nature, animals compete for resources. These resources include space, food, and mates.
Animals use aggression as a way to capture or defend these resources, which can improve their
chances of survival and mating. Aggression is a forceful behavior meant to overpower
opponents that are competing for the same resource. The outcome (victory or defeat) depends
on several factors. In insects, the bigger individuals often win. However, if two opponents are
the same size, other factors can influence outcomes. For example, an individual with more
experience may defeat an individual with less experience. Also individuals that are fighting to
gain something necessary for their survival have a strong drive, or motivation, to defeat other
individuals.
Researchers Andrew, Ken, and John study how the brain works to regulate behavior when
motivation is present. They wanted to know if specific chemicals in the brain influenced the
outcome of a physically aggressive competition. Andrew, Ken, and John read a lot papers
written by other scientists, and learned that there was a chemical that played an important role
in regulating aggressive behavior. This chemical compound, called serotonin is found in the
brains of all animals, including humans. Even a small amount of this chemical can make a big
impact on aggressive behavior, and perhaps the outcome of competition.
Picture 1. Two stalk-eyed flies rearing/extending forearms in battle. Photo credit: Sam Cotton.
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The researchers decided to do an experiment to
test what happens with increasing serotonin levels
in the brain. They used stalk-eyed flies in their
experiment. Stalk-eyed flies have eyes on the ends
of stalks that stick out from the sides of their heads
(Pictures 1 & 2). They thought that brain serotonin
levels in stalk-eyed flies would influence their
aggressive behaviors in battle and therefore impact
the outcome of competition. If their hypothesis is
true, they predicted that increasing the brain
serotonin in a stalk-eyed fly would make it more
likely to use aggressive behaviors, and flies that
used more aggressive behaviors would be more
Picture 2. A male stalk-eyed fly compared
likely to win. Battling flies use high-intensity
to the size of a dime. Photo credit: Andrew
aggressive attacks like jumping on or striking an
Bubak, June 2016.
opponent. They also use less aggressive
behaviors like flexing their front legs or rearing up on their hind legs.
To test their hypothesis, the researchers set up a fair test. A fair test is a way to control an
experiment by only changing one piece of the experiment at a time. By changing only one
variable, scientists can determine if that change caused the differences they see. Since larger
flies tend to win fights, the flies were all matched up with another fly that was the same size.
This acted as an experimental control for size, and made it possible to look at only the impact of
serotonin levels on aggression. The scientists also controlled for the age of the flies and made
sure they had a similar environment since the time they were born. The experiment had 20 trials
with a different pair of flies in each. In each trial, one fly received corn mixed with a dose of
serotonin, while another fly received plain corn as a control. That way, both flies received corn to
eat, but only one received serotonin.
The two flies were then placed in a fighting arena and starved for 12 hours to increase their
motivation to fight over food. Next, food was placed in the center of the arena, but only enough
for one fly! The researchers observed the flies, recording various behaviors of each opponent.
They recorded three types of behaviors. High intensity behaviors were when the fighting flies
touched one another. Low-intensity behaviors were when the flies did not come in contact with
each other, for example jump attacks, swipes, and lunges. The last behavior type was retreating
from the fight. Flies that retreated fewer times than their opponent were declared the winners.
After the battles, the researchers killed the flies and collected their brains. They then measured
the concentration of serotonin in each fly’s brain.
Scientific Question: How does serotonin level affect
aggressive behavior and, therefore, the probability of
winning against an opponent of similar size?
What is the hypothesis? Find the hypothesis in the
Research Background and underline it. A hypothesis is a
proposed explanation for an observation, which can then
be tested with experimentation or other types of studies.
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Check for Understanding: After reading the introduction, students should be familiar with
the following vocabulary:
• Aggression – using forceful behaviors meant to overpower opponents competing for the
same resource
• Outcome – after an aggressive act, an individual either wins (victory) or loses (defeat).
• Motivation – the drive to gain something necessary for survival
• Serotonin – a type of neurotransmitter found in all animal brains. May be responsible for
regulating aggression.
• Stalk eyed fly – flies with eyes on the ends of stalks that protrude from the sides of their
heads
• Fair test – is a way to control an experiment by only changing one piece of the
experiment at a time
Students should also be able to answer the following questions:
• Why did both flies receive corn the day before the battle? Why couldn’t you just give
one fly the corn with serotonin?
• Why did the researchers put only enough food into the trial for one fly?
• Why were the flies in the trials matched based on their size?
Teacher Note: You can stop here with your students to show them a video of stalk-eyed
flies in trial battle. The video was filmed during the experiment by the researchers listed in
this Data Nugget! It lasts 22 seconds and has no sound: https://youtu.be/LIx69mnPIQM
While watching the video, have students consider the following questions:
1. Which fly showed more aggressive behaviors? How did you decide (what is your
evidence)?
2. How many retreats did each fly make?
3. If this video segment was the entire fight, which fly would the scientists say won the
battle?
Explain how this video segment is scientifically a fair test. Is there anything you would
change to make the test results reflect aggression level even more accurately?
Answers might include:
• It is a fair test because they are size-matched, same age, regulated exposure to
food/environment, and only one has been given serotonin-rich food.
• To be more accurate, the scientists could determine winners based on
types/frequencies of aggressive behavior rather than only retreats.
Check for Understanding: Before moving on to the data, stop here to check that students
understand serotonin levels are concentrations. This means they are a ratio of something in
something else. See the note below Table 1 describing the units (pg/𝜇g). The concentration
of serotonin in the brain is determined by several factors – the amount of serotonin the fly
ingested when eating the experimental (corn mixed with a dose of serotonin) or control (corn
with no serotonin) food, the natural level of serotonin in the brain, the size of the fly, and the
rate that the fly’s body processes the chemical.
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Scientific Data:
Use the data in the following two tables to answer the scientific question:
Table 1. Serotonin Levels vs. Outcomes of Stalk-Eyed Flies
Serotonin Levels (concentration measured
in picograms of serotonin per microgram of
brain matter [pg/𝜇g])
Battle
Number
Battle 1
Battle 2
Battle 3
Battle 4
Battle 5
Battle 6
Battle 7
Battle 8
Battle 9
Battle 10
Battle 11
Battle 12
Battle 13
Battle 14
Battle 15
Battle 16
Battle 17
Battle 18
Battle 19
Battle 20
Average
serotonin
level (pg/ug)
Winner (pg/𝜇g)
Loser (pg/𝜇g)
62
190
34
57
99
23
139
67
80
121
42
49
19
69
75
89
46
97
151
21
45
38
113
24
59
32
21
16
26
26
15
22
16
29
24
21
38
36
24
106
76
37
vs.
The units used by the researchers are picograms (pg) and micrograms (𝜇g). A picogram is onetrillionth (1/1012) of a gram and a microgram is one-millionth (1/106) of a gram. The level of
serotonin found in the brain is given using the ratio of serotonin measured in picograms to brain
matter in micrograms.
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Teacher Note: For the data in Table 1, you can have students calculate standard error (SE)
or standard deviation (SD) and add error bars to their graphs to deepen this discussion.
Standard Deviation is a measurement of variability within a population. Error bars based on
standard deviation give us information on the amount of variation in the data. They can be
used to draw attention to large or small amounts of variation around the mean. Standard
error is the SD divided by the square root of the study’s sample size (SE=SD/√n). Unlike SD,
SE reflects the uncertainty in our estimate of the mean. The larger our sample size and the
less variation in the data, the more confident we can be in our estimate of the mean. Upper
error bars are calculated by adding one SE or SD to the mean, and lower bars are calculated
by subtracting one SE or SD from the mean.
Table 2. Stalk-Eyed Fly Behaviors vs. Outcomes In Battle
Observed
How many winners How many losers did
Behaviors In
did this?
this?
Battle
High-Intensity
16
5
Swipe/lunge
11
4
Jump Attack
11
2
Retreats
2
20
High-intensity behaviors include any behavior where the flies came in contact with each other.
Low-intensity behaviors included swipe/lunge and jump attacks.
Data for serotonin levels of the winners and losers are listed in Table 1. As mentioned before,
the researchers fed one of the two stalk-eyed flies serotonin-rich food before each trial. They did
this to make sure the difference in serotonin between the two flies was high enough to be
measured and have an effect on behavior. However, there were times where the natural level of
serotonin in the control fly was higher than that of the treated fly. Therefore, the data in Table 1
compares serotonin levels for winners and losers, but does NOT identify whether a fly was
treated or not. Table 2 shows frequencies of behaviors compared to outcome.
What data will you graph to answer the question?
Table 1:
Independent variable: average serotonin level (pg/𝜇g)
Dependent variable: outcome of battle (winner, loser)
Table 2:
Independent variable: observed behaviors in battle
Dependent variable: number of time behavior performed by winners
and losers (aka. # of trials with the behavior present)
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Teacher Copy, Level 2
Draw your graphs below:
Outcome of Battle
Graph 1. Average Serotonin Levels vs. Outcomes of Battles
76
Winner
37
Loser
0
10
20
30
40
50
60
70
80
Average serotonin (measured in picograms of serotonin per
microgram of brain matter)
Number of trials with behavior
present
Graph 2. Frequencies of Behavior Types By Outcome of Battles
22
20
18
16
14
12
10
8
6
4
2
0
20
16
11
11
Winners
Losers
5
4
2
High-Intensity
Swipe/lunge
Jump Attack
2
Retreats
Observed behaviors in battle
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Interpret the data:
Make a claim that answers the scientific question.
During a fight, the stalk-eyed flies with a higher serotonin
level had a higher probability of winning. The winners of the
battle showed more aggressive behaviors and did not retreat
during the fight.
What evidence was used to write your claim? Reference specific parts of the table or
graph.
Graph 1 shows that the average serotonin level of the winners
was much higher than the losers. Winners on average had twice as
much serotonin compared to losers (76 compared to 37 picograms
of serotonin per micrograms of brain matter). Graph 2 shows that
out of the 20 battles, winners made high-intensity moves in 16
of the battles, where losers only made these moves in 5 of the
battles. Winners also made more low-intensity swipe/lunge (11)
and jump attack (11) moves than did losers (4 and 2,
respectively). Graph 2 also shows that out of the 20 battles,
winners retreated in only 2 battles, while losers retreated in
all 20 battles.
Explain your reasoning and why the evidence supports your claim. Connect the data
back to what you learned about how the chemicals in the brain could influence animal
behavior and who wins in a fight.
When all other factors are held constant (the fair test
controlled for size), the chemicals in the brain of the stalkeyed fly help determine the outcome of a fight. Flies with
higher concentrations of serotonin in their brains were more
likely to be the victors in battle. These same flies were more
likely to perform high-intensity and lower intensity aggressive
behaviors. In addition, the winners rarely retreated from their
opponent, and caused their opponents to retreat in 20 of the 20
battles. The concentration of serotonin in the brain influences
fly behavior, which then determines the outcome of a fight.
What does the data from this study tell us about the scientist’s hypothesis?
The data support Andrew, Ken, and John’s hypothesis that brain
serotonin levels in stalk-eyed flies would influence their
behavior in battle and therefore the outcome of competition.
While serotonin is not the only mechanism determining who will
win a fight, it plays a major role – when a fly had higher
serotonin concentrations than its opponent, it doubled the
chances that it would win the battle.
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Teacher Note: The data in Table 1 and Graph 1 show that the winners of the battles had
higher average serotonin levels (concentrations) in their brains. The average serotonin level
of a winner was double that of the loser. When examining the behaviors in Graph 2, students
should notice that the stalk-eyed flies who performed more aggressive behaviors tended to
be those that won the battles. It may be confusing for students that winning flies were those
who performed fewer retreats, when we are focusing on increased aggressive behavior.
Clarify this by asking “What is more aggressive, retreating or not retreating? What kinds of
behaviors would likely cause an opponent to retreat?”
It is also interesting to note for students that not every battle had a winner whose serotonin
levels were higher than their opponents. There were three battles out of twenty where the fly
with lower serotonin concentrations won. It would be a good point to have students
brainstorm alternative hypotheses (other mechanisms) that could determine the outcome of
a battle. For example, the hunger level of the fly (necessity for the food) or other brain
chemicals could affect how hard the fly fought. Even though the flies were all starved for 24
hours prior to the battle, some individuals may respond differently to this increased hunger.
These three trials also demonstrate that brain chemistry is not absolute in predicting the
results of a fight.
Your next steps as a scientist: Science is an ongoing process. What new question do
you think should be investigated? What future data should be collected to answer this
question?
Student responses in this section will vary. Please read the
following Teacher Notes box for class discussion ideas.
Teacher Notes: As you segue into this section, emphasize that science is an ongoing
process, and experiments occur in a series that build upon and test each other. Just as the
scientists in this study used the scientific literature to develop their hypothesis, students can
build upon the findings of this study with their own questions. Use the following situations as
foundations for your conversation.
As a class, students can brainstorm their questions and generate new hypotheses. Here are
a few talking points to get the discussion started:
• What would we learn if the sample size (# of trials) was larger?
• If a few trials did not follow predictions while most did, what should we conclude about
our data? What does it mean that 3 of the 20 battles did not align with the hypothesis?
• Can we use our data to predict the outcome of a battle between flies? For example, if we
have a known level of serotonin for two flies, can we know who will win before the battle
takes place?
• Is a pattern the same as certainty?
• Is there certainty in science?
• How could they change this experiment to yield more reliable data?
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Teacher Notes (continued): Have students consider the importance of this research. What
useful information did we learn? Why should we care about serotonin concentrations in the
brains of stalk-eyed flies?
• At a basic level, these scientists want to understand how changes in brain chemistry
affect insect aggression and who wins in a fight. In this way, the results of this
experiment help reveal how the world works, and what explains the patterns we see in
nature; it expands human knowledge.
• These results can also be applied to humans. Flies offer a simple, ethical model to study
the mechanisms underlying aggression. Insects are the product of millions of years of
evolution, including adaptations with regard to the working of the fly brain. The fly brain is
a nice model to study the mechanisms that mediate social behaviors on a brain that is
complex, but much less complex than a human brain.
• Finally, we should remember that these neurochemicals (in this experiment, serotonin)
are found in the brains of all animal species. The physiological machinery that forms the
foundation of all animal behavior, including that of humans, is shared across the animal
kingdom. Because we share a common evolutionary ancestor, all of these mechanisms
are very similar. For example the genes and neural receptors for serotonin are
recognizably similar (nearly identical) between humans and flies. Because of these
similarities, researchers can use animal models such as flies, fish, rodents, and primates
to study physiological mechanisms underlying behavior that can eventually be applied to
humans.
What could students propose as a follow-up experiment?
• Here you could remind students that the scientists made it a fair test by controlling for
size of the flies. In addition, they also controlled for eyestalk length, fly age (same
number of weeks old, all reached sexual maturity) and similar upbringing (temperature,
humidity, diet, # of mates, and territory size). This will aid developing students’ thinking
about all that is required to control for one variable. What of these other variables might
influence the outcome of a battle, and why?
• Students might come up with the idea to battle flies of different sizes. One experiment the
researchers did as a follow up to this experiment was called “David and Goliath”. In this
experiment they used size-mismatched flies. The smaller fly was treated with serotonin
(and some left untreated as controls), similar to the experiment above. The larger fly was
left untreated. A similar arena was used to allow the flies to engage in battle. The smaller
treated flies engaged in more high-intensity behaviors than the smaller untreated flies.
However, serotonin was not enough to overcome the difference in size; the probability of
a small fly winning the battle remained low, no matter if they had high or low serotonin
levels.
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Extension Activity: Below is an excerpt from the original article discussing the experiment
and conclusions:
“High concentrations of 5-HT [serotonin] were also positively associated with
the expression of high intensity behaviors, striking with forelegs or jumping on
opponent’s dorsal side. We demonstrated that individuals who had higher
brain 5-HT concentrations than their opponent performed these high-intensity
behaviors at a much higher frequency when compared to individuals with
lower brain 5-HT concentrations than their opponent. Furthermore, the
individuals who performed high-intensity behaviors more frequently had a
much higher probability of winning the contests. The much more rare behavior
of the two, jump attack, seemed to be the most energetically costly and was
predominantly performed by the winners (over a 5-fold increase compared to
losers). Additionally, it is important to note that the losers that engaged in a
high-intensity behavior fought an opponent that performed more or an equal
number of these actions, suggesting that the frequency at which these
behaviors are expressed may serve as a predictor for winning an aggressive
bout and may be modulated by brain 5-HT activity.”
In 1-2 sentences, summarize what these researchers learned during this experiment!
The citation for the article, as well as a PDF, can be found here:
Bubak, A.N., K.J. Renner, and J.G. Swallow. 2014. Heightened serotonin influences
contest outcome and enhances expression of high-intensity aggressive behaviors.
Behavioral Brain Research 259: 137-142.
http://datanuggets.org/wp-content/uploads/2016/08/Bubaketal2014BBR.pdf
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