Introductions

There have been many studies done on the effects of alcohol on human aggression but
not so much on the crayfish. Crayfishes serve as a model to study the effects of alcohol and
aggression because they are natural territorial animals. They will fight anyone that comes into
their territory. We were able to see their aggressive behavior before and after ethanol exposure
and measure their aggressive dominance, righting response, and tail flip behavior. For our
experiment, the crayfish were given a large dose of ethanol. They were then tested with a
stimulus crayfish that acted as a control.
Based on previous studies done on other species, there is enough evidence that ethanol
leads to an aggressive behavior. Zebrafishes were given acute alcohol exposure in Echevarria,
D.J., et al 2010s study where they discovered a change in biting behavior. Even a small dose of
alcohol can have an effect on ones behavior. Furthermore, Almedia et al 19--s study showed
that a self-administration of ethanol showed an unusual increase in attack bites in rats.
Studies have found that even moderate consumptions of ethanol has an affect on the
nervous system. More specifically, ethanol affects the function of GABA-A receptors (Eckardt,
et. al). Ethanol acts like an indirect GABA agonist by binding to the GABA-A receptors and
decrease neuronal signaling. (Scripps article) Specific GABA-A receptor subtypes are sensitive
to ethanol at certain doses. There has been supporting evidence that ethanols effect on the
GABA-A receptors have some impact on aggressive behaviors. When rats were given
flumazenil, a GABA-A antagonist (??), it decreased ethanol-related aggression. (Kumar, ----)
Based on the surmounting evidence that supports the fact that ethanol may lead to a more
aggressive behavior, crayfishes given a large dose of alcohol will increase their aggressive
dominance, decrease righting response and increase the number of tail flips. Ethanol will affect
the crayfishs GABA-A receptors just like it was shown in the other studies and thus lead to an
aggressive behavior and tail flip increase. Tail flips are an way for the crayfish to charge forward
to their opponent which means an aggressive behavior would increased the tail flips shown. The
duration of a righting response would decrease because an intoxicated crayfish may have a
harder time getting back on its claws.
Methods
I. Subject:
For this experiment, we used 18 crayfishes, species name Procambarus clarkii, where
half of them were females and the other half were males. Special care was taken into
consideration when dealing with these crayfishes. They were fed brine shrimp once a week and
their waters were changed once a week as well. Each of them were put into a separate plastic
container on a 12 hr light and 12 hr dark cycle.
II. Design:
The design of our study was to measure the crayfishes behavior at baseline, administer
the ethanol and then measure the outcomes. This design lead to a some potential confounds such
as history, testing and instrumentation. History could have been a confound because someone
handling the crayfishes before could have sprayed a chemical on them that might have affected
our outcome. Testing was a confound because we saw evidence of sensitization and habituation.
(??) Lastly, instrumentation was a confound because the experimenters might have gotten better
at rating the aggressive dominance behaviors in week 2 compared to in week 1. Our quasiindependent variable was sex and our dependent variable was the level of aggressive dominance,
number of righting response and tailflips.

III. Materials:
During the experiment, the crayfishes were kept in 33.2x20.0x10.5cm plastic container.
They were in a neutral arena with the dimensions, 33.2x20.0x10.5cm and in a drug exposure
tank of 19.5x11.0x12.5cm. Materials used included gloves, bands, ethanol, graduated cylinder,
day-old tap water, 4 plastic containers, 1 metal container, a squirt bottle and a stopwatch. There
was a table of the level of aggression used as a reference. Levels 4 and 5 were omitted due to the
fact that these crayfishes were banded which would it make it impossible for them to grab or pull
on each other.
IV. Procedure:
The crayfishes were tested twice a day on Tues, Wed, and Thurs each week for 2 weeks.
For the first week of testing, the experimental Crayfish group were put in tap water for an hour.
They were then taken out and were put into the metal arena with the stimulus crayfish. The
stimulus crayfish was used to see if the experimental crayfish showed any aggressive behavior
towards it. (in discussion section?) This was done 3 times for 2 minutes each with a 2 minute ITI.
(do i need to define it?) The scores were determined by taking the maximum level of aggressive
behavior every 20 seconds for the whole 2 minutes. The 6 scores were then added up and
recorded on a spreadsheet. Two of the experimenters watched and rated the experimental
crayfish while two other experimenters rated the stimulus crayfish. We let the crayfish sit in its
cage for one minute to rest from the fighting. Next, the experimental crayfish was put on its back
to see how long it took for it get on its claws. This was the righting response that was also done
in 3 trials with a 1 minute ITI. After this test, the experimental crayfish was put back in its cage
for one minute. (?) After this minute, we squirted 10 squirts of water at the tail of the crayfish to
see if it would exhibit a tail flip. This was done 3 times with 1 minute ITI. In the second week of
testing, we administered a large dose (434 Mm) of ethanol for one hour to the experimental
crayfish. More specifically, they were put in a container filled with 25.340ml of ethanol and
974.66ml of water. The control group was the stimulus crayfish that was not given any ethanol
exposure and just in safe tap water. After the hour of ethanol exposure, they were tested in the
same conditions like in week 1. The data were then all recorded and logged on a spreadsheet.
The quasi-independent variable which was the sex of the crayfish was the between
subjects. Within subjects were the conditions the crayfish had to be in which was ethanol or tap
water. We used an intruder paradigm (??) We had the stimulus crayfish be the same across tests
to maintain consistently and selected to not treat it with ethanol. In addition, we had same sex
triads because female crayfishes tend to lose against a male crayfish. The experimenters were not
blinded from the study because they chose the details of the experiment.

Table 1. Crayfish ethogram code used to score fight intensity levels.

V. Stats
We performed a 2x2 mixed design where the between factor is sex(male or female) and
within factor is dose of alcohol (434mM or 0mM). We analyzed from the aggressive dominance
data, right data, and tailflips. Using two factor ANOVA with repeated measures one factor, we
calculated the inter-rater reliability or Pearsons r for all three dependent variables. for
habituation and sensitization as well.
Results
Dominance:
Before analyzing the data for aggressive dominance, the scores were averaged across
trials, lab sections, and observers in order to get a single number for each crayfish. A two factor
ANOVA with repeated measures on one factor with sex as between factor and alcohol treatment
as within was used to analyze the aggressive dominance data. No main effect of sex,
F(1,10)=2.83, p>0.12. No main effect of alcohol, F(1,10)=1.24, p>0.29. No main effect of
interaction between sex and alcohol treatment, F(1,10)=0.31, p>0.59. Then we got all the means
and standard errors for all four conditions: female-alcohol, female-water, male-alcohol, and
male-water. The plotted data points are shown in Figure 1. In addition, a 2 factor within ANOVA
was done to test for habituation and sensitization. The dominance scores were all averaged
across trials and lab sections in water conditions only. No significant effect of lab, F(5,55)=0.21,
p>0.95. A significant effect of trials, F(2,22)=5.38, p<0.05. No significant effect of lab and trials
interactions, F(10,110)=1.47, p>0.10. Next, the means and standard error for the trials were
plotted as shown in Figure 2. Figure 2 shows a graph where habituation occurred within trials
and not across lab sections. Pearsons r was used to calculate the correlation for between
stimulus crayfish behavior and test crayfish in tap water condition, alcohol condition, and
overall. The results were r(70)=0.02, p>0.80. Correlation was also used to examine inter-rater
reliability for both stimulus and experimental observers in both conditions. For the stimulus
observer, r(10)=0.98, p<0.0001 and for the experimental observer, r(22)=0.98, p<0.0001.

Righting Latency:
The scores for all the righting latency were averaged across trials, lab sections, and
observers to get a single score for each crayfish. A two factor mixed design where sex as
between factor and alcohol treatment as within was used. No main effect of sex, F(1,10)=0.33,
p>0.58. No main effect of alcohol, F(1,10)=1.76, p>0.21. No main effect of interaction between
sex and alcohol treatment, F(1.10)=0.05, p>0.83. The mean and standard error for all four
conditions: female-alcohol, female-water, male-alcohol, male-water were plotted and graphed in
Figure 3.
Tail Flips:
The datas for the tail flip responses were all averaged across trials, lab sections, and
observers to calculate a single score for each crayfish. A two factor mixed design where sex as
between factor and alcohol treatment as within was used. No main effect of sex, F(1,10)=1.48,
p>0.25. No main effect of alcohol treatment, F(1,10)=1, p>0.34. No main effect of interaction
between sex and alcohol, F(1,10)=0, p>1.00. There was no significant difference due to sex,
alcohol treatment and their interaction. The mean and standard error for all four conditions:
female-alcohol, female-water, male-alcohol, male-water were plotted and graphed in Figure 4.
Discussion