crayfish and ethanol: the behavioral effects of ethanol on crayfish behavior

Running head: PAUL KIM

The Effects of Ethanol on Crayfish Behavior

Neuroscience 101L – Winter 2014

Paul P. Kim

University of California, Los Angeles

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PAUL KIM

ABSTRACT

This report examines experimental findings of alcohol-related behavioral changes in crayfish of

the species P. Clarkii. Ethanol is the most widely used psychoactive drug in the world because it invokes

a myriad of effects in the human CNS. It also happens to be very pharmacologically active in other

organisms as well, including both vertebrates and invertebrates, and cause a number of neurochemical

effects. In particular, we studied the effects that alcohol has on the aggression and/or dominance

display patterns in crayfish. Crayfish that were exposed to an ethanol solution were placed in a mock

arena against another crayfish that had not been exposed to ethanol and their aggressive behaviors

were rated on a standardized scale. Other smaller scale experiments were also conducted that

attempted to replicate earlier findings regarding crayfish and alcohol-induced behavior changes. In our

data, we found no significant differences in the behaviors of crayfish that had received ethanol as

opposed to those that didn’t. This points toward the possibility that ethanol has no effect on aggression

patterns in crayfish of P. clarkii.

INTRODUCTION

We present a design of the elucidation of the effects that ethanol may have on the behavior of

crayfish (P. clarkii). In humans, prolonged ethanol use was linked to decreased gray matter volume in the

maturing brain (Nagel et al. 2005) as well as having profound effects on various cerebral and cerebellar

structures (Crews et al. 2006). As seen from the firsthand experiences of many college students at

campuses across the country, ethanol seemingly “slows” people down – reaction times decrease,

judgment is impaired, motor processes are uncoordinated, et cetera. Santhakumar et al. (2007) found

that it mediates and expresses these effects because ethanol is a CNS depressant – primarily a positive

allosteric modulator of endogenous GABA receptors. GABA receptors physiologically function as neural

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inhibitors – generating IPSCs (inhibitory post-synaptic currents) through the influx of chloride anions that

hinder the formation and propagation of excitatory signals (Santhakumar et al. 2007). Thus, by positively

modulating the activity of these receptors, ethanol is able to influence the external presentation of

increased inhibition in the brain, known as a state of intoxication (Gage 1965). In crayfish, ethanol has

been shown to affect hemolymph ethanol concentrations, and these signs of intoxication are manifested

as changes in various behavioral signs exhibited by the crayfish, particularly increased righting reflex

times and decreases in likelihood of a tail-flip escape (Friedman et al. 1988).

In this study, we examine the effects that ethanol has on dominance displays in crayfish. Earlier

experiments on crayfish showed decreases in tail-flip escapes after exposure to ethanol for a prolonged

period of time (Friedman et al. 1988). This could suggest that ethanol causes the crayfish to feel less

threat from its surroundings and promote retaliation against threats as opposed to retreat. In the

context of this experiment, this effect would manifest as increased aggression in the crayfish exposed to

ethanol: crayfish incubated in an ethanol solution should present intensified dominance displays against

other crayfish it encounters in an artificial arena. In simpler terms, our hypothesis is that ethanol does

affect behavior while our null is that ethanol does not show significant behavioral manifestations

between groups differentially exposed to ethanol.

The report begins with descriptions and explanations of the experimental design. Following up is

tabular presentations and graphical visualizations of the data obtained from the experiments as well as

the results of statistical analysis that will determine whether any differences present, if any, are

significant. The report concludes with a verbal summary of the findings of this experiment.

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METHODS

Subjects

Crayfish of P. clarkii were collected via a third-party supplier and housed in the lab in standard

shoe-box size plastic bins with the dimensions of 34.6 cm x 21 cm x 12.4 cm. The bins were filled with

two metric litres of fresh water, layered with a thin layer of gravel/small rocks to form the floor of the

bin, fitted with perforated lids to allow adequate oxygen circulation, and subjected to a repeating

schedule of 12 hours of light followed by 12 hours of dark. For feeding, the crayfish were fed once a day

with a custom mixture of shrimp in brine. A total of 18 crayfish were collected for the study, consisting

of nine males and nine females. The crayfish were then divided into six groups – three all-male groups

and three all-female groups – resulting in three crayfish per group. Within the three per group, one

served as the stimulus crayfish (untreated) and the other two were the test crayfish that received either

solution A or B the first week and the other solution during the second week.

Procedure

The experiment was conducted blind/double-blind and the design of the experiment was a

hybrid of between- and within-subjects design. Sex of the crayfish was assigned as the factor of the

between-subjects design and exposure to the ethanol solution was the factor for the within-subjects

design. The test group was incubated for one hour in an acrylic chamber (measuring 11 cm x 19.5 cm x

12.5 cm) that contained a 434 mM solution of ethanol in water prior to the beginning of the

experiments while the control group was kept in treated tap water. The experiments were also

conducted with a cross-over with each of the groups receiving the opposite treatment during the second

week compared to the first week. Whether the effects of the type of treatment during the first week

carries over and affects the data collected the second week is not known.

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To prevent the influence of intruder paradigm during the dominance display tests between the

crayfish, the trials were held in a “cage” foreign to both crayfish – the “cage” being another clear-walled

plastic bin (with the same dimensions as the housing units) lined with a thin layer of gravel at the

bottom and filled with two litres of treated tap water. The stimulus crayfish was also held constant

throughout the dominance display trials; the same, untreated crayfish of the same sex was used in each

dominance display trial. Each dominance display trial was also conducted between individuals of the

same sex to prevent the possible influence of sex in determining dominance.

The dominance display trials were conducted in the previously mentioned neutral cage. Three

trials were conducted for each test/stimulus crayfish pair. Each trial was held for two minutes each and

there was also a resting period of five minutes between each trial (inter-trial intermission or “ITI”).

Scores were assigned by three viewers every 15 seconds based on the level of aggression displayed (Fig.

1 - below) and the cumulative score was given as the final result for the trial for each crayfish for each

viewer. If the crayfish became excessively aggressive and a real threat was present, the crayfish were

then separated and the trial was terminated early. In this scenario, the maximum aggression score was

given for each time marker for the remainder of the trial.

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Figure 1 The following table is an ethogram that was used to quantitatively measure the behavior of the crayfish in regards to aggression. Each viewer recorded a score every 15 seconds for the duration of the 2 min. trial and the cumulative score was used as the aggression score of each crayfish during the dominance display trials.Intensity Level Description

-2 Tailflip away from opponent or fast retreat.-1 Retreat by slowly backing away from opponent.0 Visually ignore opponent with no response or threat display.1 Approach without a threat display, walking slowly toward the opponent2 Approach with meral spread threat display with the major chelae; antennal (2nd antennae) whips are

present, often with maxillipeds creating currents. Antennules (1st antennae) often are seen flicking.3 Initial major chela use by boxing, pushing, and/or touching with closed chelae. Chelae are not used to grasp

but can be opened and pushed. Antennal whips are more vigorous. Antennule (1st antennae) flicking is not seen.

4 Active major chela use by grabbing and/or holding opponent. Crayfish will try to turn opponents over or physically manipulate them, generating force through active major chela use.

5 Unrestrained fighting by pulling at opponent’s claws or body parts. Opponents try to pull or tear legs, antennae, or major chelae off of individuals.

The righting response trials were conducted in a large, walled metal pan measuring 40 cm x 30.5

cm x 6 cm in dimension. Three trials were conducted for each crayfish with each trial lasting up to a

maximum of two minutes. The ITI for these trials was one minute between each trial. The data collected

was the time it took for the crayfish to right and correctly orient itself after being placed in the pan on its

back. If the crayfish could not do so within two minutes, the trial was terminated and the time was listed

as two minutes.

The tail-flip response trials were conducted in the home unit of each crayfish. Three trials were

conducted for each of the test crayfish with each trial consisting of 10 firm strokes to the tail of the

crayfish using a small paintbrush. If the stroke elicited a tail-flip escape response, one point was given. If

it did not, no points were given. The ITI for these trials was two minutes between each trial. The final

data collected from these trials is the total number of tail-flips that were elicited from the crayfish.

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Statistics

The analysis of the data collected was conducted using a variety of statistical tests that allowed

us to statistically correct for variable behavior as well as see if the data obtained can be considered

significant. An ANCOVA allowed us to statistically control for the variable behavior of the crayfish

because the behavior of the stimulus crayfish could directly affect the response from the test crayfish.

Pearson’s r allowed us to determine whether the subjective, variable responses of the viewers that

judged the dominance displays were consistent in statistical variance.

To test for sensitization or habituation of the crayfish towards the effects of ethanol, a 2-factor

ANOVA F-test was done within subjects: days and the trials within the days. By doing so, we saw

whether there was a significant effect of days, trials within days, and the individual interactions between

the crayfish (whether learning/acclimation toward each other affected the results).

Because the dominance scores given to the crayfish were from two different individuals within

our group of investigators and judgment of behavior is inherently subjective, we used Pearson’s r to

determine the reliability of the scoring of each viewer and see whether the scoring was consistent for

the viewers across all the crayfish. The r value we obtained is the correlation between the two variables

(scores of viewer 1 and 2) and it would then tell us the numerical index of linear relatedness between

the two variables. In other words, it would tell us how well the two scores of each viewer for each

crayfish match up with each other and whether scoring was consistent for both viewers. The r^2 value

we calculated then tells us how much of the actual variance present in the data could be explained by

the linear relationship between the two factors.

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Finally, all three sub-experiments (dominance display, righting latency response, tail-flip

response) and the data obtained from them was analyzed using a 2-factor mixed design ANOVA with

repeated measures on one factor. The first factor was sex and this was the independent factor (between

subjects). The second factor was treatment (either solution A or solution B) and this was the repeated

measure (within subjects). These tests also allowed us to see the differential effect of ethanol between

males and females. The three F values (and their corresponding P values) then showed whether any

patterns/differences/nuances in the data could be considered as any significant when testing whether

ethanol affects the behavior of crayfish.

RESULTS

Presentation

For the data for the dominance displays (aggression scores), our analysis included two steps. In

the first step, we determined the reliability and consistency of scoring between the two raters. In the

second step, we analyzed the scores to determine whether the differences of crayfish behavior when

exposed to each solution were and could be considered as significant data. We found that our inter-

rater reliability was excellent (ie. scoring was consistent between both viewers for all crayfish) (Fig. 2 -

below) and this was determined by first averaging ratings across the three trials (of both weeks for each

crayfish for each solution they were exposed to) for each viewer. The four groups of ratings (Tuesday

rater 1 and 2, Wednesday rater 1 and 2) were then analyzed using Pearson’s r to determine the

reliability of scoring between the two raters on each day for each crayfish. Scores from the Tuesday

raters had r = 0.9713 and r^2 = 0.9434. Scores from the Wednesday raters had r = 0.9926 and r^2 =

0.9853. These scores then assure us that the judgment of the two raters is highly correlated and highly

consistent with each other.

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Figure 2 The table to the left shows a concise, tabular view of the Pearson’s r values for the dominance scores presented by both raters of each day. r = 0 corresponds to zero correlation while r = 1 corresponds to perfect correlation between the two raters. Significant data is denoted in red.

For the ANCOVA results (Fig. 3), we observed F(1,9) = 0.9, corresponding to p > 0.30. Because p

> 0.05, there was no evidence of an effect of alcohol exposure on the behavior of crayfish between the

stimulus group and test groups. However, r(10) = 0.60, corresponding to p < 0.05. This highlights a

significant correlation in the stimulus crayfish dominance scores to the test crayfish dominance scores.

Figure 3 The table below presents the ANCOVA results for the dominance displays; a between subjects comparison of treatment A (alcohol) and treatment B (treated tap) controlling for behavior of the stimulus crayfish. The results suggest that there was no evidence of an effect of alcohol exposure. Note: Observer scores were averaged for each crayfish across trials and across days. Significant data is denoted in red.

Test Statistic DFn DFd DF Statistic Value pF 1 9 - 0.96 0.3528r - - 10 0.60 0.0392

A two-factor ANOVA was done (mixed design with sex as the between factor and treatment as

within factor) with repeated measures on one factor and the results are shown in Fig. 4 (below). In all

factors of sex, treatment type, and interaction between sex and treatment, p > 0.30. Because p > 0.05,

the variance present in the results cannot be considered significant and we accept the null hypothesis to

be true. A graphical interpretation of the data can be found in Fig. 8 on page INSERT PAGE NUMBER

HERE.

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r r^2 p Tuesday raters 0.9713 0.9434 <0.0001

Wednesday raters 0.9926 0.9853 <0.0001

PAUL KIM

Figure 4 The table below presents the ANOVA results for the dominance display trials (two-factor, repeated measures on one factor, mixed design; sex as between factor and treatment as within factor). The results suggest that there was no evidence of an effect of alcohol exposure. Note: Rater scores were averaged across trials and across raters, for both days.

Source F pSex 1.14 0.313453

Treatment 0.38 0.552874Sex + Treatment 0.82 0.388779

We also sought after evidence of habituation/sensitization in the crayfish. We examined both

types of treatment, separately investigating days and trials within days. As shown by Fig. 5 (below), all p

values were p > 0.23 and thus our data did not show evidence of habituation or sensitization occurring

amongst our subjects across the various trials and days.

Figure 5 The table below presents the ANOVA results for habituation/sensitization analysis (two-factor, repeated measures on one factor, single design; days and trials both being within factors). The results suggest that there was no evidence of habituation and/or sensitization. Note: Values were based off dominance display values.

Source F pAcross days 1.4025 0.261309Across trials 1.5574 0.235179

Across days and trials 0.922 0.485717

For the righting latency trials, the average latency time was taken across the trials, followed by

an average across the days within the week. Likewise, a two-factor ANOVA was done (mixed design with

sex as the between factor and treatment as within factor) with repeated measures on one factor and

the results are shown in Fig. 6 (below). In all factors of sex, treatment type, and interaction between sex

and treatment, p > 0.10. Because p > 0.05, the variance present in the results cannot be considered

significant and we accept the null hypothesis to be true. A graphical interpretation of the data can be

found in Fig. 9 on page INSERT PAGE NUMBER HERE.

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Figure 6 The table below presents the ANOVA results for the righting latency trials (two-factor, repeated measures on one factor, mixed design; sex as between factor and treatment as within factor). The results suggest that there was no evidence of an effect of alcohol exposure. Note: Latency scores were averaged across trials and across raters, for both days.



For the tail-flip response trials, the average number of tail-flips induced was taken across the

trials, followed by an average across the days within the week. Likewise, a two-factor ANOVA was done

(mixed design with sex as the between factor and treatment as within factor) with repeated measures

on one factor and the results are shown in Fig. 7 (below). In all factors of sex, treatment type, and

interaction between sex and treatment, p > 0.14. Because p > 0.05, the variance present in the results

cannot be considered significant and we accept the null hypothesis to be true. A graphical interpretation

of the data can be found in Fig. 10 on page INSERT PAGE NUMBER HERE.

Figure 7 The table below presents the ANOVA results for the tail-flip response trials (two-factor, repeated measures on one factor, mixed design; sex as between factor and treatment as within factor). The results suggest that there was no evidence of an effect of alcohol exposure. Note: Tail-flip scores were averaged across trials and across raters, for both days.



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Graphs

Alc Tap012345678

Mean Dominance Score with Treatment

MaleFemale

Treatment Type

Mea

n Do

min

ance

Sco

re

Figure 8 The graph above shows the mean dominance scores given to each sex of crayfish depending on which type of treatment they received. Male data is represented by the blue line while female data is represented by the red line. Error bars for the male data are denoted by the lines closed by diamonds and error bars for the female data are denoted by the lines closed by circles. Error bars represent the standard error of the mean. Alc is the abbreviation for the ethanol solution and Tap is the abbreviation for the treated tap water.

Alc Tap02468

101214161820

Mean Righting Latency with Treatment

MaleFemale

Treatment Type

Mea

n Ri

ghtin

g La

tenc

y (s

ec)

Figure 9 The graph above shows the mean righting latency scores (in seconds) given to each sex of crayfish depending on which type of treatment they received. Male data is represented by the blue line while female data is represented by the red line. Error bars for the male data are denoted by the lines closed by diamonds and error bars for the female data are denoted

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by the lines closed by circles. Error bars represent the standard error of the mean. Alc is the abbreviation for the ethanol solution and Tap is the abbreviation for the treated tap water.

Alc Tap0

0.20.40.60.8

11.21.41.61.8

2

Mean Tail-flips with Treatment

MaleFemale

Treatment Type

Mea

n Ta

il-fli

ps

Figure 10 The graph above shows the mean tail-flip response scores given to each sex of crayfish depending on which type of treatment they received. Male data is represented by the blue line while female data is represented by the red line. Error bars for the male data are denoted by the lines closed by diamonds and error bars for the female data are denoted by the lines closed by circles. Error bars represent the standard error of the mean. Alc is the abbreviation for the ethanol solution and Tap is the abbreviation for the treated tap water.

DISCUSSION

Through the data that resulted from our experiments, our evidence strongly suggests that we

accept the null hypothesis and that neither alcohol nor sex (nor both) exhibit any significant behavioral

changes in aggression/dominance patterns in crayfish of P Clarkii. Almost of the p values that we

determined from our experiments were well over our threshold of p < 0.05, and thus signify that any

changes we may see have a high chance of being a false positive. However, the one correlation we did

find is a significant relationship between the dominance scores of the stimulus crayfish and test crayfish.

Aggressive or passive behavior exhibited by the stimulus crayfish was shown to be reflected in similar

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manners in the test crayfish. This could possibly be explained by the fact that the test crayfish may

respond similarly to aggression in an attempt to defend itself, and not necessarily incite aggression

randomly.

The other dependent variables we tested (righting latency and tail-flip responses) was likewise

shown to be not affected by alcohol. Because all of the p values we obtained were well over our

threshold of p < 0.05, our evidence strongly suggests that we may accept the null hypothesis. This,

however, is not coincidental with the results of prior experiments on crayfish. In normal physiological

conditions, when placed on their backs, crayfish naturally right themselves within seconds by

manipulating their appendages (Kivivuori 1977). In various experiments by Friedman et al. (1988)

however, the results strongly showed neurochemical effects in crayfish that behaviorally manifested in

anesthesia and intoxication that lead to eliminations of the righting response and tail-flip response in

high doses of ethanol. A key difference between our procedures and those of Friedman et al. (1988),

however, is that the crayfish in that experiment were incubated in an ethanol solution for several

hours/days while ours was only incubated for one hour.

Besides the discrepancies in incubation times, another factor that could be improved on in our

experiments is the extent to which the crayfish are allowed to fight. In our experiments, once the

crayfish had gotten to a point of forcefully trying to tear limbs off, they were separated and the

remainder of the trial was terminated early. A possible alternative is to band the claws of the crayfish

together so that they pose no real or significant harm to the other crayfish in their attempts to fight.

One caveat to this is that we would have to redefine the boundary at which the crayfish are merely

pushing each other and the case where crayfish attempt to pull off limbs in normal conditions (if the

claws weren’t banded).

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To summarize, the biggest factor in determining aggression and/or dominance displays in our

crayfish was not ethanol exposure, but rather conditional and dependent on the aggression/activity of

the other nearby crayfish. A vast majority of our p values allow us to support this postulation and our

evidence point towards the conclusion that we may accept the null hypothesis, that aggressive behavior

in P. clarkii is not significantly affected by alcohol.

REFERENCES

Crews FT, Mdzinarishvili A, Kim D, He J, Nixon K (2006). Neurogenesis in adolescent brain is potentlyinhibited by ethanol. Neuroscience. 137(2):437–45. [PubMed: 16289890]

Friedman RN, Bittner GD, Blundon JA (1988). Electrophysiological and behavioral effects of ethanol on crayfish. Journal of Pharmacology and Experimental Therapeutics. 246: 125-131.

Gage PW (1965). The effect of methyl, ethyl, and n-propyl alcohol on neuromuscular transmission in the rat. Journal of Pharmacology and Experiment Therapeutics. 150:236-243.

Kivivuori L (1977). Temperature acclimation of the motor activity in the crayfish Astacus astacus L. Freshwater Crayfish. 3:265-274.

Nagel BJ, Schweinsburg AD, Phan V, Tapert SF (2005). Reduced hippocampal volume among adolescents with alcohol use disorders without psychiatric comorbidity. Psychiatry Res. 139(3):181–90. [PubMed: 16054344]

Santhakumar V, Wallner M, Otis TS (2007). Ethanol acts directly on extrasynaptic subtypes of GABAA receptors to increase tonic inhibition. Alcohol. 41(3):211-21. [PubMed: 17591544]

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crayfish and ethanol: the behavioral effects of ethanol on crayfish behavior

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