1) The study examined how subordination affects crayfish behavior in mirror tests. Small crayfish were observed before and after being dominated by a larger crayfish competitor. 2) An ethogram with 8 behaviors was used to record the small crayfish's behavior over two 15-minute observation periods - one before competing and one after being subordinate. 3) Statistical analysis using a sign test found no significant changes in the crayfish's behaviors from before to after subordination. The results could not confirm that subordination affects crayfish mirror test behavior.

1. Effects of Subordination on Ornectes Virilis
Performance in Mirror Tests
Darbi O’Brien
University of Michigan Biological Station
EEB 390, Evolution
August 13, 2014
Dr. Stephen Pruett-Jones
Keywords:
aggression, agonistic behavior, mirror test, crayfish, ethogram

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Abstract
Agonistic behavior is a tool used to create fluctuating social hierarchies through levels of
domination and subordination. Procurement of critical resources like food, shelter, and mating
opportunities are settled through these aggressive social encounters. Crayfish especially have
been studied for their frequent ritualistic agonistic behavior. Although well-studied animals,
mirror tests of crayfish are not common. In this study, crayfish mirror test behavior was
compared before and after subordination. Crayfish were observed using an ethogram with 8
possible behaviors pre-competition and post-competition with a larger opponent. After an
acclimation period, a smaller individual was observed in a tank with one mirrored wall for 15
minutes. A larger opponent was introduced into the tank and the animals engaged for 15
minutes to ensure subordination over the focal individual. The opponent was removed and the
original animal was observed with the mirrored wall again. Analysis of the data revealed
insignificant results; the behaviors did not meaningfully change from before subordination to
after. Correlation between animal size and frequency of behavior was not significant. These
results could be explained through the reliance of crayfish on chemoreception, incomplete
visual separation, or the experiment restrictions.

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Introduction
Agonistic behavior and aggression has been documented thoroughly because it is such
an integral part of the animal kingdom and humans alone are subtle about these behaviors.
What is the purpose of aggression in animal behavior? Agonistic encounters aid in the
collection and control over resources such as food, shelter and mates that are important parts
of an animal’s survival (Smith 1974). In some animals, the accumulation of these contests
establishes social hierarchies of dominance which play a role in distribution and division of
resources (Dugatkin & Earley 2004). Intrinsic factors like relevant physical traits and extrinsic
effects like winner/loser effects combine to determine the outcome of contests (Dugatkin &
Earley 2004; Daws, Grills, Konzen, & Moore 2002). There are three main variables of intrinsic
factors: resource holding potential, resource value, and aggressiveness (Hurd 2006;
Dissanayake, Galloway, & Jones 2009). RHP is the sum of the characteristics of an individual
that influence its likelihood of winning contests; subjective resource value (motivation) is the
relative need or desire for a particular resource to the individual (Dissanayake et al 2009).
Aggression is trickier. Some say that aggression can only exist under assumptions that cannot
exist outside of a computer model and it is likely that aggressiveness is “more likely to
represent long-term differenced in RV” (Hurd 2006). Others say that aggressiveness should be
treated as its own variable (Dissanayake et al 2009). RHP can be quantified by measuring overall
body size, weaponry, and fight strategies, as well as available energy stores (Briffa 2008). RHP is
evaluated in all three assessment strategies: self-assessment, cumulative assessment, and
mutual assessment (Arnott & Elwood 2009; Briffa 2008). In self-assessment models, an
individual only has knowledge of their own abilities and retreat is determined solely by the

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energetic threshold, but opponents do not inflict cost. Cumulative assessment models combine
the information from self-assessment with the addition of opponent cost infliction. Higher
quality individuals inflict costs at higher rates and poor-quality individuals can endure fewer
costs and these traits affect individual RHP. Mutual assessment is unique in that opponents
compare their RHP to the other and the loser of this preliminary assessment does not have to
endure cost infliction and can retreat early on before reaching its energy threshold (Arnott &
Elwood 2009).
Behavioral experiment data are traditionally collected using an ethogram. Ethograms
break down observational periods into smaller sections and the frequency of predetermined
behaviors are recorded for each section of time. This experiment borrows the behaviors from
an experiment conducted at Brock University. In the Mercier & May (2010) experiment, crayfish
behaviors were observed during a mirror test. The subjects had either been socially stimulated
or isolated and were given the choice of two types of environment: a matte half of a tank and a
reflective half (Fig 1). The behaviors were counted and the side that behavior took place was
recorded. In this study, the behaviors used in the Mercier & May (2010) experiment were used
with the addition of a “mirror-facing” behavior because the tank design differed. This
experiment is unique from the Brock University study because behaviors both before and after
facilitated subordination of the focal animal were recorded to determine if subordination has
an effect on the behavior. The null hypothesis for this experiment is the animal does not alter
behavior patterns with a mirror test after enforced subordination. The alternative hypothesis
for the study is the animal does alter behavior patterns with a mirror test after enforced
subordination.

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Methods
Animals
Male and female crayfish, Orconectes virilis, were collected from Maple Bay on Burt Lake in
Pellston, Michigan. The animals were netted from the substrate and placed in a bucket. The
crayfish were held at the University of Michigan Biological Station Lakeside Lab. The animals’
carapace sizes were measured in milimeters and recorded, as well as their sex. Animals were
physically isolated in individual plastic containers and placed into one of two 100 gallon
aquariums. The aquariums were filled with water from Douglas Lake and each had several
tubes feeding oxygen. The crayfish were fed Hikari Crab Cuisine. All animals were isolated for at
least one week before testing in order to eliminate winner/loser effects from previous
interactions (Karavanich & Atema 1998; Bergman et al. 2003). This study had 56 total animals.
There were 19 females and 37 males. The average carapace size of females was 41.25 mm. The
average carapace size of males was 37.75 mm.
Mirror Tests
All experimentation was conducted in a glass ten gallon aquarium with approximately 60
centimeters of water. An acryllic divider with a mirror on one side was used during acclimation
and trials. A small male crayfish was selected as the test subject in each trial. The test subject’s
carapace size was recorded and the animal was placed in the tank with the divider in place at
the halfway mark of the length of the tank (Fig 2). The mirror attached the divider was facing
away from the subject. The animal was allowed to acclimate for a 15 minute period. After the

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acclimation period, the divider was turned around so the mirror faced the focal crayfish. A
competitor at least 10 mm larger than the test subject was chosen and placed in the other half
of the tank to acclimate (Fig. 2). The behavior of the test subject was monitered using the
created ethogram and eight behaviors. After the 15 minute observation period, the divider was
removed and the animals were allowed to interact (Fig. 3). Over a 15 minute encounter, the
dominance of the competitor was established through agonistic competitions. If the competitor
did not establish dominance over this period, the trial was terminated. Following the 15 minute
interaction the divider was replaced with the mirror facing the focal crayfish and the
competitor was removed. The behavior of the animal was monitered once again for 15 minutes
using the ethogram. Following each trial, the animals were placed back in their individual
containers and placed into one of the 100 gallon tanks. Every animal used in a trial was not
tested again for at least one week in order to eliminate effects from the conducted trial. 20
trials were conducted.
Ethogram
For the mirror tests, behavior of the focal crayfish was monitored using an experimental
paradigm with 8 behaviors. The observational period was 15 minutes, each minute divided into
four 15 second time slots. During each time slot, every behavior exhibited was recorded. The 8
behaviors used were cornering, facing the corner of the tank with tips of claws touching
different walls (>5 seconds); end-facing, facing the wall at one end of the tank with the tip of
one claw touching the end wall; turning, changing the walking path direction from clockwise to
counterclockwize or vice versa; crossing, leaving the perimeter of the aquarium and walking at

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least one body length to any other wall; reverse-walking, walking backwards for at least one
body length; freezing, abrupt cessation of all visible movement including antennae (>5
seconds); rearing-up,standing on fourth and fifth walking legs while lifting the thorax against
the wall and placing at least one leg on the wall; and mirror facing, facing the mirror with the
tip of one claw touching the mirror. All behaviors and definitions were taken from the study at
Brock University, excluding mirror facing. Two ethograms were created from each trial, pre-
competition and post-competition. Each of the behaviors was totaled from each ethogram.
Analysis
The total times the behavior was performed pre-competition was compared to post-
competition behavior totals, which requires a nonparametric test for paired data. After totaling,
a sign test was performed for each behavior to compare observed behavior of the animal to the
expected behavior. The differences in behavior totals was catgorized as either positive or
negative. If a behavior occurred more in the pre-competition observation than the post-
competition, the behavior was counted as “+”, 1 in the binary code created for data analysis. If
a behavior occurred more in the post-competition observation than the pre-competition, the
behavior was counted as “-”, 0 in the binary code. This data was used in the sign test formula:
The p-values were then examined closer to determine statistical significance. A linear
regression analysis was also performed to determine correlation between carapace size and
frequency of behaviors.

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Results
Sign Tests
Results of the sign tests for each behavior proved to be insignificant. The sign tests were
performed to determine if the crayfish behavior considerably strayed from the expected half
negative, half positive continuous distribution of two random variables. However, the results
showed that the behavior did not stray significantly from the expected mean of 0 between
positive and negative test results. The p-values ranged from 0.4119 to 0.1153 and none were
significant (Table 1). Cornering, end-facing, and reverse-walking had p-values of 0.4119.
Crossing, rearing up, and mirror-facing all had p-value=0.2517. Turning and freezing had the
lowest p-values with 0.1153. All 8 behaviors had insignificant sign test results.
Linear Regression Tests
Linear regression analyses were performed to determine corrlation between size and frequency
of behaviors. Results of these tests were insignificant as well. Reverse-walking behavior had
R2=0.0092 (Fig 4). Cornering behavior had an R2=0.0234 (Fig 5). Freezing behavior had
R2=0.0646 (Fig 6). End-facing behavior had the least significance with R2=0.003 (Fig 7). Rearing
up behavior had R2=0.0482 (Fig 8). Mirror-facing behavior had the most significant R2 with
0.1046 (Fig 9). No significant results came of this analysis.
Discussion

9. O’Brien 9
Subordination did not produce significant changes in behavior in the before and after
competition mirror tests. The null hypothesis was accepted because there was no quantifiable
change in behavior. There are several reasons this could have happened. This study was
conducted over three weeks with a sample size of 56 animals and only 20 trials. The small
sample size, number of trials and short experiment conduction time all could have affected the
results of the experiment. Another reason for these results is the lack of complete visual
isolation during the focal individual’s pre-competition mirror test and the acclimation period for
the opponent. In the experimental tank, the crayfish were isolated with both in a different half.
However, the individuals were not completely visually isolated because the mirror did not
extend the entire length of the acryllic divider (Fig 10). In the Brock University experiment, the
methods stressed the importance of complete isolation in order to demonstrate the
importance of visual inputs for crayfish social interaction (Mercier & May, 2010). Perhaps the
focal animal seeing his opponent through the clear acryllic sections that were not covered by
the mirror affected his behavior during the pre-competition mirror test.
An indepth look at crayfish communication provides an alternative reason the experiment did
not yield significant results. Numerous experiments (Wolf & Moore 2002; Moore & Grills 1999;
Cook & Moore 2008; Aquiloni & Gherardi 2010) have shown that crayfish rely heavily upon
chemoreception in social interaction and searching for resources. Crayfish have chemosensory
organs that they utilize to “read” smells in the water and the location of the smells (Moore &
Grills 1999). This ability is translated into agonistic behavior and mate selection, individuals
reacting to smells of other individuals. Therein lies a flaw with the experimental design of this
study. Because chemoreception is such a vital component of social interaction of crayfish, there

10. O’Brien 10
is a possible override of the assumption that crayfish will interact with a reflection the same as
another individual. The animals used in this experiment were allowed to interact with a mirror
with the theory that the animal will register its reflection as an opponent. However, the
individual would not be getting a chemical read of the supposed opponent so the facilitated
behavior would not be comprable to a real interaction of two individuals. It is possible that the
post-competition behavior would be abetted by the remaining scent of the opponent from the
competition period. The Brock University experiment was designed to test the effects of
isolation for mirror testing and did not operate on the premise that the crayfish would treat the
reflection as another individual (Mercier & May 2010). Further experimentation with a larger
sample size would be prudent to test if crayfish will recognize a reflection as another individual.
In this additional experimentation, a dominance effect would be tested as well. The same
experimental design with the opponent being smaller than the focal crayfish. Females should
be tested using this design, as well.
This study explored an experiment type uncommonly associated with crayfish. Aggression and
agonistic behavior is an integral part of crayfish social life which is easily measured. What is not
as easliy measured is the ability of crayfish to distinguish real individuals from reflections.
Although this experiment had insignificant results, mirror tests for crayfish should not be
abandoned and, on the contrary, should be investigated even further.
Acknowledgements
I would like to thank Peter Rogers for his partnership in this experiment and being an
outstanding colleague. Thanks to Stephen Pruett-Jones for assisstance on this project, as well as

11. O’Brien 11
Paul Moore for being a constant fountain of crayfish knowledge. Thank you Kelle Urban,
Samantha Antczak, Sarah Wofford, David Edwards, Michelle Busch, Ana Jurcak, Paul, and PJ for
aid in collecting animals.

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Figure Legends
Figure 1. An illustration of the experimental tank for the Mercier & May (2010) experiment at
Brock University.
Figure 2. An illustration of the acclimation period and tank setup for the focal animal of this
study.
Figure 3. An illustration of the pre-competition mirror test and opponent acclimation tank setup
for this study.
Figure 4. An illustration of the competition tank setup for this study.
Table 1. The results of the sign tests conducted for each trial and their respective p-values.
Figure 5. A graphical representation of the correlation of reverse walking behavior instances to
the size of the animal tested, for both before and after competition.
Figure 6. A graphical representation of the correlation of cornering behavior instances to the
size of the animal tested, for both before and after competition.
Figure 7. A graphical representation of the correlation of freezing behavior instances to the size
of the animal tested, for both before and after competition.
Figure 8. A graphical representation of the correlation of end facing behavior instances to the
size of the animal tested, for both before and after competition.
Figure 9. A graphical representation of the correlation of rearing up behavior instances to the
size of the animal tested, for both before and after competition.
Figure 10. A graphical representation of the correlation of mirror facing behavior instances to
the size of the animal tested, for both before and after competition.

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Figure 11. An illustration of the possible incomplete visual isolation during the pre-competition
mirror test.

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Figures and Tables
Figure 1

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Figure 2

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Figure 3

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Figure 4

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Table 1

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Figure 5
y = -0.0351x + 5.7815
R² = 0.0007
y = -0.0616x + 4.9528
R² = 0.0092
0
2
4
6
8
10
12
14
16
18
20
20 25 30 35 40 45
InstancesofBehavior
Size
ReverseWalking
Before
After
Linear (Before)
Linear (After)

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Figure 6
y = 0.0917x + 7.2978
R² = 0.0028
y = -0.3228x + 21.747
R² = 0.0234
0
5
10
15
20
25
30
35
40
20 25 30 35 40 45
InstancesofBehavior
Size
Cornering
Before
After
Linear (Before)
Linear (After)

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Figure 7
y = -0.6348x + 36.202
R² = 0.0393
y = 0.8221x - 14.298
R² = 0.0646
0
5
10
15
20
25
30
35
40
45
50
20 25 30 35 40 45
InstancesofBehavior
Size
Freezing
Before
After
Linear (Before)
Linear (After)

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Figure 8
y = 0.2336x + 3.1611
R² = 0.0122
y = -0.1292x + 15.721
R² = 0.003
0
5
10
15
20
25
30
35
40
20 25 30 35 40 45
InstancesofBehavior
Size
End Facing
Before
After
Linear (Before)
Linear (After)

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Figure 9
y = -0.2894x + 15.976
R² = 0.0453
y = -0.3512x + 18.485
R² = 0.0482
0
5
10
15
20
25
20 25 30 35 40 45
InstancesofBehavior
Size
Rearing Up
Before
After
Linear (Before)
Linear (After)

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Figure 10
y = -0.5713x + 28.682
R² = 0.1046
y = -0.0583x + 8.9314
R² = 0.0009
0
5
10
15
20
25
30
20 25 30 35 40 45
InstancesofBehavior
Size
Mirror Facing
Before
After
Linear (Before)
Linear (After)

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Figure 11

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Literature Cited
Arnott, G., & Elwood, R.W. (2009). Assessment of fighting ability in animal contests. Animal
Behaviour. 77(5):991–1004.
Aquiloni, L., & Gherardi, F. (2010). Crayfish females eavesdrop on fighting males and use smell
and sight to recognize the identity of the winner. Animal Behaviour. 79(2):265-269.
Briffa, M. (2008). Decisions during fights in the house cricket, Acheta domesticus: mutual or
self-assessment of energy, weapons and size? Animal Behaviour. 75(3):1053-1062.
Cook, M.E., & Moore P.A. (2008). The effects of the herbicide Metolachlor on agonistic
behavior in the crayfish Orconectes rusticus. Archives of Environmental Contamination
and Toxicology. 55(1):94-102
Daws, A.G., Grills, J., Konzen, K., & Moore, P.A. (2002). Previous experiences alter the
outcome of aggressive interactions between males in the crayfish, Procambarus clarkii.
Marine and Freshwater Behaviour and Physiology. 35(3):139-148.
Dissanayake, A., Galloway, T.S., & Jones, M.B. (2009). Physiological condition and
intraspecific agonistic behavior in Carcinus maenus (Crustacea: Decapoda). Journal of
Experimental Marine Biology and Ecology. 375(1):57-63.
Dugatkin, L.A., & Earley, R.L. (2004). Individual recognition, dominance hierarchies and
winner and loser effects. Proceedings of the Royal Society, Biological Sciences, Series B.
271(1547):1537-1540.
Enquist, M., Leimar, O., Ljungberg, T., Mallner, Y., & Segerdahl, N. (1990). A test of sequential

27. O’Brien 27
assessment game: fighting in the cichlid fish. Nannacara anomala. Animal Behaviour.
1990(40):1-14.
Hurd, P. (2006). Resource holding potential, subjective resource value, and game theoretical
models of aggressiveness signaling. Journal of Theoretical Biology. 241(3):639-648.
Mercier, A. Joffre, and Holly Y. May. "Recording Behavioral Responses to Reflection in
Crayfish." Journal of Visualized Experiments 39 (2010): Web.
Moore, P.A., & Grills, J. (1999). Chemical orientation to food by the crayfish, Orconectes
rusticus: Influence of hydrodynamics. Animal Behavior. 58(5):953-963.
Smith, J.M. (1974). The theory of games and the evolution of animal conflicts. Journal of
Theoretical Biology. 47(1):209-221.
Wolf, M.C., & Moore, P.A. (2002). Effects of the herbicide Metolachlor on the perception of
chemical stimuli by Orconectes rusticus. Journal of the North American Benthological
Society. 21(3):457-467.