1. START
Stress Effects On The A-maze-ing Crayfish
Sally Le and Thanh Thuy Le
Clayton State University, Department of Natural Sciences
INTRODUCTION
METHODS
CONCLUSIONS
1. Five crayfish were selected from communal tanks to use throughout the
experiment.
2. Individual crayfish is placed in the dark maze map for 15 minutes to explore. Red
lighting is used to observe and record.
3. An aversive zone is designed in which the aversive zone is kept dark while the
remaining areas are illuminated. Unstressed crayfish are given 15 minutes to
explore the new maze map to serve as a control.
4. Unstressed crayfish are tested again on the same maze map and are given 15
minutes to explore; however, if the crayfish cross into the aversive zone, they will
be electrically shocked to induce aversion.
5. These crayfish will undergo the same procedures in step 4 so that each individual
will be conditioned to avoid that area due to fear and anxiety.
6. These conditioned crayfish are then tested again in the same aversive maze map.
However, the crayfish are shocked in a separate chamber before they are given 15
minutes to explore. Crayfish under stress would most likely avoid the light areas
and remain in the dark regions. Both stressed and unstressed crayfish have a
higher preference for the dark regions1. With this, their memory will be tested.
7. Each trial is recorded and observed for path tracing and number of turns (left vs.
right) based on the crayfish preferences. An outline of each crayfish’s route
traveled will be digitally designed similar to the D/L+ maze in the article1.
Based on the locomotion pattern observed from the crayfish, stressful conditions
contribute to a decrease in locomotion. Meanwhile, observations in the ratios of
light/dark indicate signs of memory retention. Although time in the light did not
restore back to control levels, the ratios for 2 out of 3 crayfish maintain preference for
the light after the induced aversion in the dark. Furthermore, in recordings of the
same two out of three crayfish, they are noted to pause at the “shock” zone as well as
one retreating from the zone before eventually entering it. However, there cannot be
any definitive conclusions made about how stress affected the crayfish memories in
this experiment simply due to the low amount of subjects surviving to the final stage.
In further studies, it would be best to use a large number of subjects to ensure the
data pool is large enough to represent the behaviors of most crayfish.
1. Fossat, B.C., et al. (2014). Anxiety-like Behavior in Crayfish is Controlled by Serotonin. 1293-1297.
2. Mirk, S., Wermcrantz, B. (2007). Caffeine Inhibits Serotonin's Enhancement of EJP Amplitude in
Crayfish Deep Extensor Muscle. 21-22.
3. Musolf, B.E., Spitzer, N., Antonsen, B.L., Edwards, D.H. (2006) Serotonergic Modulation of
Crayfish Hindgut. Biol. Bull. 217, 50-64.
4. Tierney, A. J., Andrews, K. (2012). Spatial Behavior in Male and Female Crayfish (Orconectes
rusticus): Learning Strategies and Memory Duration. 23-33.
REFERENCES
RESULTS
Drawing ideas from the similarities between how crayfish and humans handle
stress and anxiety, we decided to test the learning capabilities of crayfish under
stressed and unstressed conditions. The induced circumstances are similar to that of
a college student predisposed to stress to recall information during exams, the
stressed crayfish are observed whether they could retain memories of aversive zones.
Through conducting this experiment, the results can possibly relate how stress can
affect memory.
Based on the effects of stressor and environmental lights effects on crayfish
anxiety as well as the correlation between anxiety levels on depressing exploring
behavior, the stressed crayfish will remember the induced aversive zone and will be
less inclined to enter it even if it is favorably darkened.
Fig. 5 – Dark (Control)
RESULTS
ACKNOWLEDGEMENTS
Thank you to Dr. Musolf, Dr. Braun, Justin Cropsey, and the crayfish for all their help
in making this experiment possible.
START
START
Observing the control trials such as the one
shown in figure 5, an aversive zone was designed. The
darkened region, shown in dark grey, is the area that
each crayfish tend to cross the most. The figure to the
left display the path trace of one unstressed crayfish,
▲, use as a control to observe locomotion in the
light/dark map.
Fig. 6 – Light/Dark (Control)
The figure to the left display path trace of one
crayfish, ▲, through the dark maze which serves as a
control for observing locomotion. Observations are
used to determine which area each crayfish spends the
most time in or repeatedly return to in order to
designate an aversive zone at a high traffic region.
Fig. 1 – Dark
START
After inducing aversion to the crayfish using the
maze map shown in figure 3, the crayfish was shocked
in a separate chamber and was allowed to explore in
the same maze map. The figure to the left display the
path trace for one stressed crayfish, ▲. Focal subject
was observed to travel less frequently and the speed
of locomotion was reduced. In addition, focal subject
tend to retreat when entering the aversive area.
Fig. 8 – Chamber  Light/DarkSTART
In the control, all three
crayfish spent roughly the
same time in the light. When
they are shocked to induce
memories of an aversive zone
in the dark, all crayfish stayed
90% in the light. Stressing the
crayfish in the chamber, 2 out
of 3 retained staying in the
light while  did not.
START START
Fig. 3 – Aversive Fig. 4 –  L/DFig. 2 – L/D
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LightTImeRatio
Light Area
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Fig. 9
Fig. 10
• Figure 1 serves as the overall control for crayfish’s locomotion. The maze is a dark
room illuminated by a red lamp.
• Figure 2 serves as a control for locomotion in light and dark zone. The maze is in a
dark room illuminated by spot lights beneath the maze.
• Figure 3 is the maze map used to shock the crayfish within the maze. The thunder
bolt indicates the aversive zone where they are shocked.
• Figure 4 is the same maze map as figure 2. Crayfish are shocked in a chamber prior
to entering.
In the control, dark was least
preferred by all three. When
they are shocked to induce
memories of an aversive zone
in the dark, all avoided the
dark. Stressing the crayfish in
the chamber, 2 out of 3
retained aversion to the dark
while  did not.
START
A path trace of one unstressed crayfish, ▲,
through the aversive maze map. Focal subject entered
the aversive zone once and was shocked in that area
to induce stress and aversion. Locomotion was
significantly reduced and an avoidance of the aversive
zone was observed.
Fig. 7 – Aversive

2. 0%
20%
40%
60%
80%
100%
120%
Control Aversive Chamber
Average In Dark Versus Light
Time in Dark Area/Total time Time in Light Area/total time
Fig. 9
In the light/dark control, unstressed crayfish spent more time exploring the
light areas. This is expected due to the fact that the illuminated portion is larger
compared to the darkened region. When focal subjects were shocked in the aversive
zone, they retreated and remained in the light portion over 90% of the time.
However, under stress, the crayfish explored both light and dark areas almost
equally.