The Role of Serotonin in the Maintenance of a

“Loser Effect” Among Crayfish Battles

By: Jannah Hicks & John Gonzalez

Lenoir Rhyne University

March 13th, 2021

Introduction

Aggressive interactions occur in almost all species. The importance of these interactions

is to determine an individual's success. (Zulandt, 2001). Many species relay information to each

other by using visual, chemical, or other sensory signals (Bergman, 2005). The winner effect is

defined as the increased probability of one individual winning a fight, following a previous

winning experience. Whereas, the losing effect is defined as the decreased probability of an

individual initiating the aggressive interaction. (Rutte, 2006). Though, winning experiences has

no influence on the probability of initiation of aggressive interactions, it has shown an increase in

the likelihood of an individual initiating attacks that specifically cause harm to the other.

Serotonin is a neurotransmitter which acts on the frontal areas of the brain in order to

inhibit the amygdala, the part of the brain that controls fear and anger. Serotonin acts a

developmental signal and plays an important role in cell regulation as it aids in cell proliferation,

apoptosis, migration, synaptogenesis, and differentiation (Çetin, 2017). Due to the various

functions of the neuromodulator, serotonin has been proven to be held accountable for many

mental health disorders, such as anxiety, autism, mood disorders, and attention-deficit

hyperactivity disorder (ADHD) (Çetin, 2017). Research has shown aggression and impulsivity is

primarily linked to serotonin. Aggression can be defined as behavior that harms or injuring

another living being who is motivated to avoid such treatment (Davies, 1987). Though serotonin

affects vertebrates and invertebrates differently (Huber, 1997), it has been shown that the

neurotransmitter serotonin has been linked to stimulate aggressive behavior in both humans and

animals.

This experiment was aimed to determine if serotonin can reverse the loser effect by

comparing aggressive interactions of crayfish before and after the losers have been injected with
serotonin. In the experiment, it was hypothesized that an increase in serotonin levels would

increase the likelihood of reversing the loser effect in crayfish resulting in the loser crayfish

winning in the next round.

Experimental Design

In this experiment, the fighting arena was prepared by filling a Tupperware container

with 1-2 inches of conditioned water with a divider in place to separate the arena into two halves.

Two same-sized crayfish of the same sex were placed into the areana and acclimated for 10

minutes. Once the acclimation period ended, the divider was removed to allow interaction and

battle between the two crayfish for a time period of twenty minutes. Using the ethogram in Table

1 of the student outline for this experiment, different behaviors expressed during the crayfish

battle were scored and recorded. After the crayfish battle, the winning crayfish were injected

with 100µl of saline and the loser crayfish were injected with a 100µl serotonin solution. Both

the saline and the serotonin solution were injected between their second and third limbs due to

efficient circulation in that area. Winners from the first battle were used as opponents for the

losers in the next battle. Due to crayfish communicating chemically as well as visually, the

fighting arenas were reset before the next battle. The serotonin injected loser and saline injected

winner were placed into the divided arena for an acclimation period of 10 minutes. The divider

was removed and the behaviors were scored and recorded once more. Further information of the

materials used in this experiment are listed in Table 2 of the Student Outline.
Table 1. Crayfish ethogram (adapted from Moore, 2007)

Results
 Once all of the battles were finished, data scores were compiled from each individual in

each match. By having the class data, averages of winners, losers, scores, etc. were able to be

calculated. As shown in Figure 1, both crayfish that were injected with saline and crayfish

injected with serotonin had a higher average ethogram score. Looking at the two different

injections alone, crayfish that were injected with serotonin had a higher average ethogram score

than crayfish that were injected with saline. However, a T-test was performed to determine if

there is a significant difference between the type of injection the crayfish received. Although, it

was found that there was no significant difference between the type of injection the crayfish

received, the data showed a trend towards serotonin and the increase of aggression as well as the

increase of the average ethogram score.

 Figure 2 compares the average scores of loser crayfish before and after the injection of

serotonin. From this graph, it is evident that the average individual scores were significantly

higher after the loser crayfish were injected with serotonin. While Figure 2 compares the average

score of loser crayfish and the time of the injection of serotonin, Figure 3 compares the average

individual score of winner crayfish before and after the injection of saline. From this graph, it is

shown that there is no significant difference between the average score of winner crayfish and

the time of the injection of saline.

 Figure 4 illustrates the average individual score after the saline injection versus the

average individual score after the serotonin injection. From this graph, it is clear that crayfish

that were injected with serotonin had an overall higher average individual score. However, there

were no significant differences found between the type of injection the crayfish received and the

average individual score.

Discussion

After analyzing and comparing the class’s data, the data collected was not statistically

significant; however, there is an obvious trend towards serotonin being able to reverse the loser

effect and increasing the aggression in crayfish. This trend supports the hypothesis that serotonin
reverses the loser effect in crayfish battles. Comparing the crayfish which were injected with

serotonin and the crayfish injected with saline, there is only a 3 point difference, implying no

significant difference. Though there is no significant difference between the two different types

of injections, Figure 2 shows that there was a significant increase in the average individual scores

after the injection of serotonin. Overall, the tests that were conducted and data that was collected

clearly showed serotonin’s effect on the aggression of crayfish.

In this experiment, there were many possible errors that could have occurred. One being

incorrectly collecting other group members’ data which could skew the results of the data. This

data may have also been influenced by incorrectly injecting the crayfish as it could have been

injected too deeply or too shallow, as well as potentially harming the crayfish. Further research

needs to be done in order to support the hypothesis collectively and not based on the trend line

alone.
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