Studying how surroundings affect Procambarus clarkii dominance through controlled observational experiments

By Ethan Hiltner, Cheyenne Shields, Robert Russo, and Sejal Hule

www.msu.edu/~hiltnere/

LB 144 Organismal Biology
Thursday 7 PM

Webpage written by The Life of Pablo Picasso
Teaching assistants: Morgan Kiryakoza and Mellissa Ungkuldee

Revised by Cheyenne Shields

Introduction

Revised by Cheyenne Shields
Crayfish inhabit nearly every body of water in North America as well as in various other countries. They are freshwater crustaceans that resemble lobster (Hunter et al., 2001). Until now there are more than 500 species of crayfish recorded (Norris et al., 2008). They are found in greater diversity in Southeastern North America. Particularly in Kentucky and Louisiana the Mississippi basin, but they also live in Europe, New Zealand, and East Asia Nearly all crayfish live in freshwater, although few survive in saltwater (Dunoyer et al., 2016). Fossilized records of crayfish have been found dating back as far as the late Paleozoic and early Mesozoic (Emory University, 2008). Crayfish have a very interesting array of dominant tendencies which are affected by one's surrounding environment and the situation being observed. Dominance is shown in a variety of situations such as territorial disputes, resource disputes, and in shows of courtship for females. Among crayfish, fighting between size-matched animals leads to an abrupt change of behavior as the new subordinate retreats and escapes from the attacks and approaches of the dominant (Issa et al, 1999). It is marked by an escalating series of behaviors leading to grappling and wrestling with the heavy claws (Bruski and Dunham, 1987; Huber and Kravitz, 1995; Krasne et al., 1997).

The sequence of tail-flip and raising claws trace the development of their dominance hierarchy (Herberholz, Fadi A. Issa and Edwards, 2001). The fighting decreases when one animal (the new subordinate) breaks off contact with its opponent (the new dominant) by escaping or retreating (Issa et al., 1999). The neural mechanisms in crayfish and lobsters that underlie this decision are unknown but may involve presence of similar genes to humans that mediate discrete behavior patterns displayed during agonistic interactions. Our study observes thoroughly the established dominance behavior of two crayfish when they are introduced to various resources and situation in the food, shelter, and bare tank (controlled) experiments.

The goal of this research is to get a better idea of what resources procambarus clarkii value most when presented with crayfish of the same species and size (±0.2 in). In addition, we looked closely for dominance characteristics that are seen in multiple scenarios. We predict that the crayfish will fight in every trial and one will be decided the first dominant in under a minute (Herberholz, et. Al., 2016). In several similar experiments of similar nature, a dominance battle began the instant two procambarus clarkii were put into the same tank. We also predict there will be multiple times that dominance is reestablished within the twenty-minute period if the two crayfish have to had a dispute in the past. Reestablishment of dominance is defined by the retreat of a crayfish while the resource in contention has been claimed by another crayfish (Bergman et. Al., 2003). We chose the 20-minute interaction period because of a similar experiment done by researchers studying resource availability and dominance relationships between two crayfish in a confined tank (Gruber et. Al., 2016). In addition, we believe that the crayfish will fight for longer and with more intensity when there is a food source available. In contrast, we believe that the crayfish will fight for less time and with less intensity when presented with a shelter source. This is because shelter sources are highly dependent on whether there are predators in the area. In our particular experimental set up there was no presence of predators to increase the intensity of dominance battles regarding shelter (Chibucos et. Al., 2015).

Understanding dominance tendencies of procambarus clarkiiP helped us better understand how and when humans are likely to display dominance. Similar to animals humans also include status and territory competition as motives that are particularly elicited by conspecific challenge situations and, when the aggression is successful, outcomes of reduction of challenge and enhancement of resource control and status. The response characteristics of human aggression have been dramatically altered by human verbal, technological, and social advancements (Blanchard et. al., 2003). Additionally, for people as well as for animals, fear of defeat or punishment is a major factor inhibiting the expression of offensive aggression (Blanchard et. al., 2003) . It is common for humans to display aggressive tendencies subconsciously. Things such as a raised voice, clenched fists, invading personal space, intense sweating, and a reddening face have been associated with anger and aggression (Changingminds.org, 2016). These subconscious cues make it possible to observe groups of people and read nonverbal signs of aggression. This was key in determining the experimental setup of our human experiment.

To dig further into the topic of aggression and how it related to dominance we looked at several genes present in both humans and crayfish that affect the function of serotonin receptors. We looked at serotonin receptors in specific because they have been found to regulate aggression and dominance/subordinate relationships in crayfish and humans. (Meier, et. Al., 2015). The gene HTR1B encodes a G-protein-coupled receptor for serotonin and is in the 5-hydroxytryptamine receptor subfamily. This particular gene is present for humans and crayfish and manages the release of dopamine, serotonin, and acetylcholine (NCBI, 2016). Function of this gene has been shown to be positively correlated with spikes in aggressive behavior tied to social dominance in humans (Boer, Koolhaas, 2005).

Methods

Revised by Ethan Hiltner
First one red swamp crayfish sized 2-4 inches, scientific name Procambarus clarkiiP, was placed in a tank, which was obtained from Lyman Briggs College, with no cover or food resources. Crayfish were purchased from Carolina Biological Supply. Each trial was between two of the three crayfish. Combinations were crayfish A and B, crayfish B and C and crayfish A and C(Figure 3.). After a 5 minute acclimatization period a second crayfish was introduced (Herberholz, et al, 2016). The crayfish were then left in the tank together for an observation period of 20 minutes (Herberholz, et al, 2016). Following the 20 minute observation period crayfish were returned using an aquarium net provided by Lyman Briggs College to their respective 5 gallon tanks. Detailed observations were recorded by hand and the entirety of the trial was video recorded on an Apple iPad. Observations were based on a crayfish ethogram code (Bergman, et al, 2003). This trial served as the positive control because researchers expected the crayfish to interact with aggression with no resources present. The experimental setup for the tank and iPad were constant across all 3 trials in this control experiment and are described in detail in Figure 4.

The next portion of our observational experiment was based on shelter. Three trials were ran for the two types of shelter resources we chose for a total of 6 observational periods. Shelter resource 1 had 22 rocks from 2-4 inches in length which were collected from Lansing River Trail in East Lansing, MI. Shelter resource 2 was a 4 inch long section of 2 inch diameter polyvinyl chloride pipe obtained from Lab C3 of Holmes Hall, East Lansing, MI. The independent variables in each trial were the shelter in the tank. These shelters were placed in the tank prior to either crayfish entering the tank. One crayfish was then introduced using an aquarium net. After a period of 5 minutes a second crayfish was introduced (Herberholz, et al, 2016). The crayfish were left in the tank together for an observation period of 20 minutes. All 6 shelter trials were video recorded on an Apple iPad for future use. Detailed observations were recorded by hand using an aggression intensity ethogram code (Bergman, et al, 2003). Each trial had the same experimental setup shown and described in Figure 4.

During the following portion of the experiment researchers introduced one Procambarus clarkii crayfish to a five gallon tank that had one of two food sources in it. 3 trials were ran for each food source for a total of 6 observational periods. This was done in order to allow all 3 crayfish interact with each other in both food experiments. Food resource 1 had 8 shrimp pellets from XTreme aquatic foods and absolutely no shelter (Seymour, 2014). Food resource 2 had 3 pieces of chicken in the tank totalling .05 pounds taken from Michigan State University Holmes Hall Cafeteria. In each trial 1 crayfish was put in the tank for a period of 5 minutes with an aquarium net to settle and become acclimated to tank conditions (Herberholz, et al, 2016). Then we added the second crayfish with an aquarium net for 20 additional minutes. We observed for any dominance behavior such as snapping claws, raising claws, and attacking in their interaction for a period of 20 minutes using the ethogram code described above(Herberholz, et al, 2016). After the 20 minutes were up the crayfish were returned to their respective tanks with an aquarium net. Experimental setup was consistent with all other trials as described in figure 4.

Following the crayfish experiment we conducted our human experiment to show how aggression is subconsciously displayed between humans in a normal friendly setting. We chose Akers MSU cafe for our human experiment setting. We observed humans during situations where we expected we would see subconscious dominance displays. We looked for the subconscious cues such as raising voices, frequently hand gestures, clenched fists, slamming things, straightened posture, and reddened face. In order to not characterize non-aggressive tendencies as such we waited to record cues until more than one was shown. Observation periods in the cafeteria included 3 trials of 20 minutes each. Aggression between humans was recorded using the back facing camera of a Microsoft Note Laptop.

In addition to recording aggression intensity using a crayfish ethogram (Figure 1) we took note of re-establishment of dominance between crayfish in each trial. A re-establishment in dominance was defined as one crayfish retreating from the other in the tank followed by a re-initiation of a dominance battle by the crayfish who originally retreated (Herberholz, et al, 2016). This data was observed through iPad footage taken of each interaction period.

Statistical Analysis

All trails were viewed by all 4 researchers through  MP4 files taken on an iPad pro. While viewing videos researchers recorded ethogram and dominance re-establishment data in their lab notebooks. After all trails were viewed and all data was recorded we performed t-tests for 2 Correlated samples to determine the difference in mean aggression intensity between resource trials and the control trials. For individual t-tests, the sum of all ethogram data for each 20 minute observational period was placed as 1 value. All sums for all food trials and shelter trials were tested against the sums of ethogram data in our control experiments. These t-tests were done with a 95% confidence interval which accounted for possible errors in the recording process. In addition, we implemented 5% error bars to figure 2 due to possible human error in the process of recording observations based on the ethogram code. All statistical analysis in this study was done using Vassar Stats

Results

Revised by Robert Russo

For all experimental results we kept the experimental setup constant (Figure 4)The control experiments served as a baseline. Observations consisted of intensity levels based off of a crayfish ethogram code (Bergman, et al, 2003) which can be seen in [Figure 1]. Intensity levels were recorded for the duration of the 20 minute trials (Figure 2). Intensity level totals for each trial are as follows. Control Trial 1 Crayfish A&B- total intensity: 47 and DR (dominance establishment): 4. Trial 2 Crayfish A&C- total intensity: 33 and DR: 3. Trial 3 Crayfish B&C- total intensity: 69 and DR: 6.

The first food trial we ran consisted of 8 shrimp pellets. All trials were video recorded and observations were recorded based off of the videos. Observations consisted of intensity levels based off of a crayfish ethogram code (Bergman, et al, 2003) which can be seen in [Figure 1]. In addition to recording ethogram data researchers also took note of every time dominance was re-established between the two crayfish (Figure 3). Intensity level totals for each trial are as follows. Shrimp Pellets Trial 1 Crayfish A&B- total intensity: 132 and DR (dominance establishment): 11. Trial 2 Crayfish A&C- total intensity: 62 and DR: 5. Trial 3 Crayfish B&C- total intensity: 89 and DR: 8. The second food trial done with chicken pieces demonstrated that crayfish do value food resources over shelter. An overall observation of the dominance behavior demonstrated by the red swamp crayfish (Procambarus clarkii) when introduced to the food  revealed that food resources have significant value and the crayfish demonstrated higher intensity of dominance establishment while claiming the resources. The intensity levels were recorded based off a crayfish ethogram code (Bergman, et al, 2003) which can be referred from [Figure 1]. All trials were video recorded and detailed notes were taken based on the observations. All trials were recorded with a 20 minutes time duration. The results of intensity level total for each trials are as follows: Chicken piece trial 2, specimen A and B- total intensity:68, DR:06, Specimen A and C- total intensity:13, DR:02 and Specimen Band C- total intensity:60, DR:05. The overall study shows that specimen B and C had the highest intensity of fighting [Figure 1]. Regardless of the intensity levels, the pursuit for claiming food resources was seen as a significant finding which provide a simple analysis for more complex behaviors potentially be impacted by other resources and surrounding.

The first shelter trial was conducted with a 4 inch long section of a 2 inch diameter polyvinyl chloride pipe (PVC). We used an ethogram presented in [Figure 1] to determine the levels of intensity shown between the crayfish (Bergman, et al, 2003). The results showed that crayfish A and C showed the highest levels of dominance. Trial 1 of 3: Crayfish A&B-Total intensity: 2 with a 0 dominance re-establishment. Trial 2 of 3: Crayfish B&C-Total intensity:1 with a 0 dominance re-establishment. Trial 3 of 3: Crayfish A&C-Total intensity:13 with 2 dominance re-establishments. The second shelter trial was conducted with about twenty-two rocks collected from Lansing River Trail in East Lansing, MI. Again this was video recorded with an iPad to be later viewed for dominance level determination using the ethogram (Bergman, et al, 2003). The results showed that crayfish A and C showed the highest levels of dominance. Trial 1 of 3: Crayfish A&B-Total intensity:1 with a 0 dominance re-establishment. Trial 2 of 3: Crayfish B&C-Total intensity:1 with a 0 dominance re-establishment. Trial 3 of 3: Crayfish A&C-Total intensity: 17 with 3 dominance re-establishments.

Several t-Tests for 2 independent samples were run. This statistical test compared the mean intensity level for each set of trials with the mean intensity level of the control set of trials. Mean value (rock shelter) - mean value(control) = -43.33. Meaning that the mean intensity level for the rock shelter was on average 43.33 points less than the average intensity level of the control trials. The confidence intervals for this particular t-test were ±64.8%.Mean value (PVC pipe shelter) - mean value(control) = -44.33. Meaning that the mean intensity level for the PVC pipe shelter trials was on average 44.33 points less than the average intensity level of the control trials. The confidence intervals for this particular t-test were ±59.6%. Mean value(chicken) - mean value (control) = -2.67. This means that the mean intensity level for the chicken trials was on average 2.67 points less than the average intensity level of the control trials. The confidence intervals for this particular t-test were ±52.68%. Mean value (shrimp pellets) - mean value (control) = 44.67. This means that the mean intensity level for the shrimp pellet trials was on average 44.67 points greater than the average intensity level of the control trials. The confidence intervals for this particular t-test were ±87.43%.

After conducting all t-test for our different experimental trials used a t-test to determine differences in the mean across the entirety of the food and shelter trials. Mean value(food trials) - Mean value (shelter trials) = 64.8333 points with a confidence interval of ±46.48% . This data indicates that that on average there was significantly more intense aggression in food trials than in shelter trials.

In the human portion of our study we found little aggression in Akers hall cafeteria. This further backed our claim that aggression in humans and crayfish is context dependant. Of the aggression cues we were looking for; face reddening, clenched fists, raised voice, intense sweating, invasion of personal space, and straightened posture; only one was shown at a time throughout the 3 groups captured on video. Of the 3 20 minute trials there were only 4 instances where a single aggression characteristic was shown. In addition, each aggression cue shown was often offset by laughing, smiling or eating. These results indicate that humans show little aggression when eating food with friends in a buffet environment.

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Figures

             Revised by Sejal Hule

Figure 1. Crayfish ethogram code used by researchers to quantify aggression intensity data (Bergman, et al, 2003). This crayfish ethogram code classifies different kinds of behavior shown by the crayfish based on the intensity levels of aggressive and submissive actions. The intensity levels are represented using a number scale and a description of said aggressive behaviors. These number ranks go from a -2 ranking for retreating behavior to 5 which is the highest intensity aggression behavior. For each 20 minute interaction period researchers marked any time one of these 8 behaviors was observed. This ethogram allowed researchers to assign quantitative values to the intensity of aggressive behaviors displayed by crayfish in each experimental trial.

Figure 2. Aggression levels of crayfish in the 5 different experimental settings Letters A, B, and C represent each crayfish being studied. For each permutation 1 crayfish was dropped in the environment for 5 minutes to acclimate before another crayfish was placed in the environment for a remainder of 20 minutes. Number totals for each 20 minute trial were collected by the researchers through ethogram analysis (Figure 1) of videos taken on an iPad pro. The total number of each permutation was the sum of all ethogram aggression numbers for both crayfish present in the tank over each particular 20 minute period. Error bars account for the presumed 5% human error in recording aggressive actions between crayfish.

Figure 3. Number of times dominance was re-established between crayfish in the 5 experimental settings. Letters A, B, and C represent each crayfish being studied. For each permutation 1 crayfish was dropped in the environment for 5 minutes to acclimate before another crayfish was placed in the environment for a remainder of 20 minutes. Over this 20 minutes the researchers took note of dominance re-establishment by observing each crayfishes particular behavior. Retreat followed by a re-initiation of contact by either crayfish in a trial was considered a re-establishment of dominance (Herberholz, et. Al, 2016).

Figure 4. Experimental Setup across all crayfish trials. The same experimental setup was used for the entirety of the trials. For all 3 control trials, all 6 food trials and all 6 shelter trials we placed the tank in the middle of the same laboratory table. An Apple iPad was then setup on a rubbermaid container for every trial so that the entire tank could be recorded for the entirety of each 20 minute interaction period. Interaction periods were done at the same time everyday in the same lighting with the same amount of people around the tank. The water in this experimental tank was replaced every new set of trials in order to keep water chemistry constant (Bergman, et al, 2003).

Figure 5. LB144 Documentary