The Wild Wombats

Observed Defense Response Behavior in Fox Squirrels in Urban and Rural Setting Correlates to Human Behavior in Danger

By: Lauren Ott, Raya Patel, & Alexis Wood

LB 144 Organismal Biology

Thursday 8:00am

Douglas Luckie, PhD, Joel Betts, Hayden Stoub, and Samantha Thacker

11/30/2016

www.msu.edu/~patelr19/

https://www.youtube.com/watch?v=g1eQqZBhsNc

Introduction

Responsible for: Lauren Ott

The study of animal behavior starts by understanding how a species functions and the body anatomy that is assimilated with its behavior. Some examples of information that spark different behaviors in species include sound, smells, hunger, terror, and danger. Some of the most noticeable behaviors in animals are feeding behaviors, mating behavior, social behavior, and communication (Cheal & Sprott, 1971). Knowing how genes and an environment unite to form a species actions is important in the field of animal behavior, because the genes of a species help determine the evolution of proceeding populations in order to help figure out a species current behavior. Many scientists study animal behavior because it closely relates to human behavior by the means of social and reproductive behavior (Nowiki & Searcy, 2005). One common behavior among all animals including human is the fight or flight response. This response becomes is very strong when the body senses any threats, danger to survive, or any attacks from predators. The generated stress leads to communication between species. Both humans and squirrels show specific responses when they are faced with an external threat. Specific behaviors become apparent when faced with a threatening or high stress situation, particularly in squirrels and humans (Milosevis, 2015).

Many different urban animals use a variety of signals as a means of communication in the environment. The fight or flight response behavior of squirrels in threatening environments has been the topic of many observational experiments. The four different squirrel behaviors that that are being studied in this experiment include quadrupedal vigilance, bipedal vigilance, tail-flicking, and tail-fagging. Quadrupedal vigilance behavior is defined as the squirrel standing on all four legs, head above shoulders, and the head is scanning the area. Bipedal vigilance behavior is defined as the squirrel is standing only on hind legs, the head is above the shoulders, and scanning the area (Unck et al., 2009). Tail flicking behavior shows that the tail moves fast, at medium power for a short period of time. Only the end portion of the tail moves while the rest of the tail stays aligned with the body. Tail flagging behavior shows the tail moving at high power, usually the tail moves above their heads. This involves the most movement of the tail (Partan et al., 2010). In many cases, it was found that when presented with a threat, squirrels may exhibit tail-flagging movement as a means to deter a predatory threat. Many squirrels have been observed to move slightly closer to the predator in order to assess the level of danger in addition to the tail flagging (Bennett et al., 1984). It is believed that tail flagging behavior can be used as way of communication to neighboring squirrels as a warning about a possible threat and as a defensive mechanism against the threat itself (Barbour & Clark, 2012).

In order to survive, most animals commonly employ fight-or-flight responses in times of need; similarly, humans, use thier physiological ability and unconscious behaviors to effectively fight threats. Humans exhibit different defense mechanisms and body behavior when faced with danger. A defense mechanism is an unconscious behavior that lowers anxiety coming from a potentially harmful situation. A defensive behavior can be one of many body language cues that humans can exhibit when faced with danger. The amygdala, located in the cerebral hemisphere, has a variety of different functions such as social communication and the expression of fear in humans. The amygdala is a critical component that assesses an environment for potential danger (Phelps & LeDoux, 2005). This is the part of the brain that is responsible for processing information and trying to decide if you should run away or stay and fight during dangerous or high stress situations. Some of the instinctive responses exhibited by humans when faced with fear include: freezing, flinching, fight, and flight. Freezing is where the subject stops all responses and movements for a period of two seconds or more. Flinching is a quick turn of the head or any quick, nervous movement of the face and body. A defensive attack or fight is when the subject responds to a threat with violence or an attack. Flight is running away from the threat (turning and moving away from the threat at a pace faster than walking). These are the defensive behaviors exhibited by humans when confronted by a threat without even being aware of them happening (Blanchard, 2008).

Being able to find a homologous behavior between an animal such as squirrels and humans is really relevant is science. They both have extensive genomes that are easily testable and could prove closeness of the two species. A gene is a unit of heredity that is transferred from a parent to offspring and is held to determine some characteristics of the offspring. Phenylethanolamine N-Methlytransferase (PNMT) is a gene that is found in humans and squirrels, and is responsible for the release of adrenaline into the bloodstream during potentially harmful situations. The PNMT gene is a protein coding gene found on chromosome 17 with 1,594 base pairs. For this gene, stress hormones activate the PNMT gene (Morita et al., 1992). The protein PNMT methylates the amine in norepinephrine by catalyzing the transfer of the methyl group from the cofactor S-adenosyl-L-methioine (SAMe) to norepinephrine, creating epinephrine. SAMe is a common substance on which enzymes act, typically involved in methyl group transfers (Mann et al., 2001). The greater the amount of stress hormones released, the greater the greater amount of epinephrine produced. The PNMT gene play a key step in regulating the epinephrine production; epinephrine is adrenaline. Adrenaline is a hormone secreted into the bloodstream by the adrenal gland, especially in conditions of high stress and fear. It causes increases rates of blood circulation, increased heart and breathing, enhancing reflexes, increase blood supply to the muscles, dilating pupils, and preparing muscle for exertion, allowing for both human and squirrels to be able to quickly respond to a threat (Kitahama et al., 1992). With this gene similarity, in the future, squirrels could possibly be used to test for diseases in humans which could lead to a healthier society. This also show evolution because differrent genomes have several similarities.

In order to better understand alertness and the "fight or flight" response in squirrels, we plan on observing squirrels in an urban, human populated environment, and a rural, naturalistic environment. We will be observing and recording the different stance positions and tail behaviors of the squirrels in the different environment. The hypothesis of this experiment is if a squirrel is surrounded by a human dense environment, then it will tend to be more alert and responsive to its surroundings and are more cautious of nearby threats than squirrels in a more natural environment, because it has been found that there is a positive relationship between squirrel wariness and the density of humans. We hypothesize that the behavior identified when squirrels are considered alert will be similar to the behavior of a human that is aware of their surroundings, this is because humans demonstrate behaviors that can be compared to those of squirrels when slightly frightened (Parker & Nilon, 2008). The objectives of this experiment are to determine if squirrels from a more populated environment are more vigilant to predators, threats, and their overall surroundings. Also, the squirrels found in a more isolated and natural environment, where there are no human interaction, are used as a control in order to see if there is a difference in actions and behavior. With these objectives we hope to support our hypothesis that squirrels in an urban environment use more visual body language and are more alert to their surroundings (Partan, 2010).

Methods

Responsible for: Raya Patel

Observational Study in Squirrels

In order to learn more about the fight or flight response in both squirrels, an observational study was conducted in two locations; a high stress environment and a low stress environment. For our squirrel experiment, the study sites were located in two different parts of campus. The first was located behind Holmes Hall, both near the bird feeder that is strictly dedicated to observing and cannot be approached by humans and in the Sanford Natural Area, the forest behind Holmes Hall, where the squirrels were observable from the blind. Observable from the blind meant that we were able to observe the squirrels without them knowing anyone was present. Observing from a blind reduces bias because the observers don't effect the behavior of the squirrels. This is a very low stress environment for squirrels because there are little to no presence of threats that will drastically and visibly alter squirrel behavior. The second observation site was located on the portion of the Red Cedar River, which lies between Shaw Hall and Wells Hall. This was a high stress location where the squirrels were surrounded by loud noises, buildings, landscapes dominated by artificial architecture, and a high human density, where human subjects are always walking to and from class and tend to sit feeding the surrounding animals (ducks and squirrels). This environment was defined as having at least three to five peoeple in a thirty foot radius of the squirrel. We are observing random squirrels that are in the area, not choosing and marking the specific squirrels. There was not an exact sample size that was sought for this study, because we were not tracking which squirrels we had already observed, so we do not have a set number of squirrels to observe. The sample size is always recorded and averaged if needed at the end of each trial. Because it is hard to tell the difference between male and female squirrels, and the different genders of the squirrels does not affect the behaviors we are studying, all squirrels are useable for observations. The materials that were used in order to carry out this experiment were iPhones, iPads, and digital cameras to get documentation the behavior of the squirrels as well as notebooks to record the results of the observations. The video feature on the iPhone was used to take video recordings of the squirrels in order to capture the observed data.

In order to observe alertness and defense responses in fox squirrels on the Michigan State University campus, we will perform an observational study. Data was collected at three times; 8am, 12pm, and 5pm in the two different locations. The date, time, and weather were all recorded in order to account for any environmental variables and minimize and error. There was a total of eighteen trials throughout the experiment (three times for each location and time) and the data was recorded in 60 second intervals for each squirrel we observed over a one hour sitting. The four behaviors that were recorded include bipedal vigilance, quadrupedal vigilance, tail flicking, and tail flagging as defined above (Partan et al., 2010). Based on these behaviors, we focused on the frequency of tail flicking and tail flagging in urban and rural environments based on the time of day. We also focused on the frequency of tail flicking versus tail flagging relating to the quadrupedal vigilance or bipedal vigilance presented by the squirrels. All observations were recorded as long as they fit the definition for the appropriate behaviors. If the tail does not fully move above their head at high power, it will not be counted as tail flagging, and if the squirrel is not fully upright on its two feel it will not be considered bipedal vigilance. Each member of the group has done extensive research as well as watched credible videos in order to recognize the correct behavior that the squirrels were exhibiting. After the observational study is completed, there will be statistical analysis done on the data to ensure the data is statistically significant. For this particular study, a chi-squared test was used; a chi squared test is applied when you have two categorical variables from one population. It is used to determine if there is significant association between the two variables. Because a chi-squared test is used with categorical data, there will be no error bars produced on the graphs. The chi squared test was used in this experiment to test the significance of the data that was collected between tail flicking and tail flagging versus quadrupedal vigilance and bipedal vigilance in squirrels. It was also used to determine the significance between time of day in each location versus the frequency of tail flicking and tail flagging behaviors (Satorra & Bentler, 2001).

Observational Study in Humans

Human subjects were also observed in order to compare fight or flight behaviors between squirrels and humans as well as alertness in high and low stress environments. When observing the people, all defensive behaviors exhibited were recorded and compared to that of a squirrel. The observations were recorded using an added stimuli, such as door slamming, a book being dropped, or a car horn honking, and also observed in a natural setting, for example crossing the street while a car is coming. Observation sessions took place at 10am, 4pm, and 10pm, each lasting an hour, in order to see if the time of day also had an effect on human alertness. The data was recorded in two minute intervals for each human subject we observed over a one hour sitting. There were eighteen trials for this experiment, three for each set time in both a low stress environment and a high stress environment. The general locations that these human subjects were observed in focused on areas where stress levels would be heightened, i.e. an empty study room, walking down a quiet street alone at night, or in an empty hallway. The human subjects were also observed in area where there is very little to no stress, such as crowded study lounges, walking down the street with a group of people, and walking through a crowded hallway. We observed and recorded behaviors such as stopping all movements and responses for two seconds (freezing), a quick turn of the head (flinching), or quick movements away from the source (flight), and a defense attack (fight)(Zhu et al., 2005). The sample size for this study was, but no limited to 15-20 humans per trial. Both male and females were observed because gender did not play a key role on what we were observing and recording. Each member of the group had done a significant amount of research to ensure they were observing and recording the correct behaviors. The materials used in this experiment were notebooks and iPhones to record the found data. After the observations were concluded, a statistical analysis of the data collected was performed using the chi-squared method explained above, because we are comparing two categorical variables from one population. The chi squared test was used in this experiment to test the significance of the data that was collected between the different human behaviors, such as flinching, freezing, flight, or flight in areas of low or high stress (Satorra & Bentler, 2001).

Gene Analysis

Primers

In order to test the presence of the Phenylethanolamine N Methlytransferase (PNMT) gene in squirrels and humans, a Polymerase Chain Reaction (PCR) test was conducted. Before the annealing process in the PCR test could occur, the correct primers were found and use for the gene amplification, in order to bind a single strand of DNA from the hair of a Fox Squirrel, obtained from WDNAS laboratories located in the UK, to hair from a human (Ogden et al, 2005). A primer has to be synthesized by the enzyme, primase, which is an RNA polymerase, before DNA replication can occur (Williams et al., 1990). Twenty base pairs were identified using the NCBI database in the human and Fox Squirrel genomes to create the primers. For the human PNMT gene, a primer that was twenty base pairs long was found, with a forward primer sequence of 5'-GCCTACCTCCGCAACAACTAC-3' and a reverse primer sequence of 5'-GGGCCTGAACCAATGTCGAT-3'. For the PNMT gene found in squirrels, a primer that was twenty base pairs long was found, with the forward sequence 5'-CCCCAGTGTCTACGTGAGGA3'-, and the reverse primer sequence of 5'-TCCTGCCACTTGCTTCTGTC-3' (NCBI, 2013). The primer was a sequence of twenty base pairs starting at 5 prime and ending at 3 prime, meaning the polymerase starts its replication at the 3 prime end of the primer, and copies the complementary strand to create the reverse polymer (Neff et al., 2002). After these primers were designed, the correct annealing temperatures in which the primers would bind were determined using the oligonucleotide properties calculator based on the specific primer strands found (Sipos et al., 2007).

DNA Purification

After the primers were identified, a DNA purification had to be performed. DNA purification is done in order to reduce or eliminate contamination on a sample (Hawkins et al., 1994). The hair samples from the squirrel and the hair sample from the human were washed and cleaned by submerging them into fresh water to remove the surface of dirt and other possible contamination. The hair sample were then picked up with sterilized forceps and washed with 500ul 70% ethanol in a 1.5ml micro centrifuge tube. The hair was kept in a tube containing sterile, deionized water. The hair samples were then further examined under a magnifying glass in order to remove any existing body fluids. The hair samples were cut off five to ten millimeters from the root end for digestion (Ghatak etal., 2013).

PCR

Once the DNA was purified, the PCR test could begin. Polymerase chain reaction or PCR is a method used to strengthen a copy of DNA, creating many copies of a particular DNA sequence (Fangan et al., 1994). PCR was performed in a 25ul reaction volume, holding a 100ng template DNA, 0.2pM of each primer, 2.5ul of the 10x PCR buffer, 1.5 mM MgCl2, 200 mM dMTPs, and 1 unit Tag of DNA polymerase. This mixture of the reaction was heated to 94 degrees Celsius for five minutes followed by 40 cycles. Each of these cycles involved one minute of denaturation, where the hydrogen bonds holding the complementary strands of DNA together are broken, at 94 degrees Celsius, one minute annealing or attaching at 63 degrees Celsius, one and a half minute extension or elongation at 72 degrees Celsius, and finally 10 minutes of elongation at 72 degrees Celsius. The PCR strengthening product was then exposed to gel electrophoresis (Ghatak et al., 2013).

Gel electrophoresis

A gel electrophoresis was created to help get a visual representation of the DNA in squirrels and humans. The one percent agarose gel was made by mixing one gram of agarose and with 10mL 10x TBE and 90mL of water. The mixture was then heated in a microwave for 2-3 minutes or until the solution was completely dissolved. After the solution cooled or until it was warm to touch, 10um of SYBR safe 3 mix was added to the solution and poured into a gel box with a gel comb placed at the negative end so that the gel could form. The gel set at room temperature for about 30 minutes to ensure the gel fully solidifies (Muyzer & Smalla, 1998). After the gel solidified, the comb was carefully removed and each of the samples were loaded into separate wells. Well 1 contained water, as a negative control, well 2 contained a standard molecular weight ladder Precision Plus Protein Dual Color Standards from Bio-Rad (Hunter et al., 2005), well 3 contained the squirrel sample, and well 4 contained the human sample. The gel was placed into the machine and enough recycled TBE was added to the chambers of the machine so that it completely covered the gel. The lid was shut and the black and red electrodes were connected to their negative and positive terminals respectively. The machine was turned on, the voltage was set for a range between 80-120 volts, and gel was run for 20-30 minutes. Once the current stopped, the gel was removed from the machine and the placed under an ultraviolet light in order to see the RNA placement. The results were observed and compare between the human and squirrel (Tenover, 1995).

Results

Responsible for: Alexis Wood

As seen in figure 1B, we predict that squirrels will be most active and alert in a human populated environment than in a natural environment because the squirrels will be more conscious when there are surrounded by constant potential threats, humans, in their surrounding environment. Squirrels have adapted to living in high stress environments by constantly watching out for nearby threats and by always being prepared to leave the area when in the presence on a threat (Partan et al., 2010). Also, based on figure 1C, we predict that squirrels will show more tail flagging while in a bipedal vigilance and tail flicking while in a quadrupedal vigilance, because squirrels tend to be in a bipedal vigilance while they are scanning their surrounding vantage point while in a quadrupedal vigilance they just tend to scavenge for food (Unck el at., 2009). For this particular study, a chi-squared test was used. The chi squared test was used in this experiment to test the significance of the data that was collected between tail flicking and tail flagging versus quadrupedal vigilance and bipedal vigilance in squirrels. It was also used to determine the significance between time of day in each location versus the frequency of tail flicking and tail flagging behaviors. We predict that the p values found from this test will be less than 0.05, because that means that data will be significant (Holt et al., 1980)

Based on figure 2B, we predict that humans will react more prominently showing distinct behaviors such as freezing, flinching, fight, and flight in less populated and quiet area because high stress environments tend to heighten the fight or flight response as the threats would be less expected, while low stress environments tend to be more crowded and loud showing a lower fight or flight response (Blanchard, 2008). A chi squared test will be performed once there is enough observed data in order to see if the data collected is statistically significant. Once the chi squared value if found, that value is used to find the p-value. If the p-value is less than 0.05 then the data is significant (Holt et al., 1980).

Based on figure 3A we predict that there will be a correlation between human behavior and squirrel behavior when they are aware of any threats in their environment, this is because the alert behavior displayed in both species by certain movements (Parker-Nilon, 2008). We believe that there will be a correlation between squirrel behavior and human behavior. We predict that the Phenylethanolamine N-Methlytransferase (PNMT) gene is present in both squirrels and humans and responsible for releasing adrenaline into the bloodstream, because adrenaline is released into the body during high stress, fight or flight situations (Kitahama et al., 1992). In order to further test the presence of the PNMT gene in human and squirrels a gene analysis was performed. Based on figure 3B, we predict that the predicted bands shown in green will be located at about 1,549 base pairs because that is the recorded weight for the PNMT gene. A chi squared test would be conducted to determine if the data was statistically significant by comparing the frequency of human and squirrel behavior with the respective environment (Holt et al., 1980).

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Predicted Figures

Responsible for: Alexis Wood

Figure 1. Predicted results of the frequency of tail flicking and tail flagging in urban and rural environments. A. This is a GIF showing the four different behaviors that we are observing in squirrels: quadrupedal vigilance, bipedal vigilance, tail flicking, and tail flagging. Quadrupedal vigilance behavior is defined as the squirrel standing on all four legs. Bipedal vigilance behavior is defined as the squirrel is standing only on hind legs (Unck et al., 2009). Tail flicking behavior shows that the tail moves fast, at medium power for a short period of time. Tail flagging behavior shows the tail waved at high power, usually the tail moves above their heads (Partan et al., 2010). B. This figure shows our predicted frequency of the two different tail behaviors of the squirrels were observed and recorded at different times of day in order to see if the tail behaviors of squirrels have an effect on the alertness of squirrels. Observations occured at 8am, 12pm, and 5pm in both an urban and rural environment. Over a span of 3 months, 18 trials were conducted, 9 for each location. Based on the figure we predict squirrels will show more tail flicking in rural environment and tail flagging in an urban environment because it is believed that the tail-flagging can be used as way of communication to neighboring squirrels as a warning about the possible threat, and as a defensive mechanism against the threat itself (Parker & Nilon). A chi squared test will be performed, comparing the time of day in each location and the frequency of the tail behavior ovserved by the squirrels once there is enough observed data in order test see if the data collected is statistically significant. Once the chi squared value is found, that value is used to find the p-value. If the p-value is less than 0.05 then the data is significant(Holt et al., 1980) C. This figure shows the predicted frequency of different tail movements based on specific vigilance in both rural and urban environments. Tail flicking and tail flagging were both observed based on either quadrupedal or bipedal vigilance while in a rural or urban environment. Observations occured at 8am, 12pm, and 5pm in both an urban and rural environment. Over a span of 3 months, 18 trials were conducted, 9 for each location. Based on the figure, we predict that squirrels will show more tail flagging while in a bipedal vigilance and tail flicking while in a quadrupedal vigilance, because squirrels tend to be in a bipedal vigilance while they are scanning their surrounding vantage point while in a quadrupedal vigilance they just tend to scavenge for food (Unck el at., 2009). A chi squared test will be performed, comparing the vigilance of the squirrel to the frequency of tail behavior observed once there is enough observed data in order test see if the data collected is statistically significant. Once the chi squared value if found, a chi squared distribution table can be used to find the p value. If the p-value is less than 0.05 then the data is significant (Holt et al., 1980).

Figure 2. Prediction of Behavior in Humans in Low and High Populated Areas. A. This GIF shows the four different behaviors that we are observing in humans: flight, flinching, freezing, and fight. Flight is running away from the threat (turning and moving away from the threat at a pace faster than walking). Flinching is a quick turn of the head or any quick, nervous movement of the face and body. Freezing is where the subject stops all responses and movements for a period of 2 seconds or more. Fight is when the subject responds to a threat with violence or an attack (Blanchard, 2008). B. This figure shows our prediction of the results of the human study observed in the two different locations. Observations occured at 10am, 4pm, and 10pm in both low stress, high populated environments and high stress, low populated environments. Over a span of 3 months, 18 trials were conducted, 9 for each location. Specific human behaviors, including freezing, flinching, fight, or flight, were observed from a distance and recorded. Based on the figures, we predict that humans will react more prominently in in less populated and quiet area because less stressful environments will heighten the fight or flight response as the threats would be less expected (Blanchard, 2008). A chi squared test will be performed once there is enough observed data in order to see if the data collected is statistically significant. Once the chi squared value if found, that value is used to find the p-value. If the p-value is less than 0.05 then the data is significant.

Figure 3. Genetic Correlation between Humans and Squirrels A. This figure show our predicted results of the gel electrophoresis in the PNMT gene on both humans and squirrels after the PCR test was conducted. The x-axis shows the different wells and the y-axis shows the different molecular weights. For the human PNMT gene, a primer that was twenty base pairs long was found, with a forward primer sequence of 5'-GCCTACCTCCGCAACAACTAC-3' and a reverse primer sequence of 5'-GGGCCTGAACCAATGTCGAT-3'. For the PNMT gene found in squirrels, a p rimer that was twenty base pairs long was found, with the forward sequence 5'-CCCCAGTGTCTACGTGAGGA3'-, and the reverse primer sequence of 5'-TCCTGCCACTTGCTTCTGTC-3' (NCBI, 2013).The molar weights decrease as they get further away from the wells because it is easier for smaller molecules to travel further. Well one contained a standard molecular weight ladder Precision Plus Protein Dual Color Standards from Bio-Rad (Hunter et al., 2005), well 2 contained the squirrel sample, and well 3 contained the human sample. The standard molecular weight ladder was used to determine the weight of the samples, determining the location of the gene. The DNA extracted from both the human and squirrel hair cycled 40 times. Each of these cycles involved 1 minute of denaturation, the hydrogen bonds holding the complementary strands of DNA together are broken, at 94 degrees Celsius, one minute annealing or attaching at 63 degrees Celsius, one and a half minute extension or elongation at 72 degrees Celsius, and finally 10 minutes of elongation at 72 degrees Celsius. The PCR strengthening product was then exposed to gel electrophoresis (Ghatak et al., 2013). The predicted bands, shown in green, are expected to be located at about 1,549 base pairs, which is the recoded weight for the PNMT gene (NCIB, 2013) B. This figure shows our predicted results of the total human and squirrel behavior frequency taken for each of the urban and rural environment. We predict that there will be a correlation between squirrel and human behavior when they sense a threat in the environment because of the behavior displayed in both species by their different movements (Parker & Nilon, 2008). PNMT is a gene present in both squirrels and humans and is responsible for the fight or flight response (Kitahana et al., 1992). A chi squared test would be conducted to determine if the data was statistically significant.

Figure 4. Observed Defense Response in Fox Squirrels in Urban and Rural Settings This is a five minute film documentary that encompasses the video capture of the defense response behavior in Fox Squirrels in an urban and rural environment correlating to human behavior in high and low stress environments.