The purpose of our study is to investigate a single homologous gene found within fox squirrels and humans and observe if the gene accounts for a certain homologous behavior. We hypothesize that the fox squirrels and humans will exhibit similar fight or flight responses when exposed to a threatening audible stimuli, which included a hawk and German shepherd vocalization, because the ADRB2 gene would be present in both species. The ADRB2 gene is a receptor that responds to Adrenaline which would be essential for fight or flight responses. The significance of our research is to replicate previous tested research methods including playback observational studies, while also implementing PCR and gel electrophoresis to observe the ADRB2 gene within fox squirrels and humans in order to determine whether or not both species express the gene behavior. The experimental design included a playback study where predatory stimuli would be elicited through a speaker to sample sizes of fox squirrels and humans. A PCR was used to amplify the ADRB2 gene and gel electrophoresis was used to analyze the base pairs. We predict that the ADRB2 gene is present in both species based on our results from observing homologous fight or flight behaviors which correlate with each other. Also from observing the ensemble project conducted by Yates and his colleagues (Yates, et al) which portrays squirrels exhibiting the ADRB2 gene.

The fox squirrel, scientifically known as Sciurus niger, are a widely distributed tree squirrel species native to North America. While the squirrel can move about with lots of agility, it lives most of its life in trees, spending only about seven hours outside of its nest each day (Wassmer, 2016). Fox Squirrels also have many potential predators that are potential dangers including hawks and foxes, (Graham, 1997). The fox squirrel values its ability to sense such danger as much as any other mammal, including humans and react to the danger accordingly. Reacting to danger would require animals to determine its fight or flight responses. The fight or flight response, in a general sense, is a chain of rapidly occurring reactions in the body that help mobilize animals in the face of threatening circumstances. It is an inherent ability possessed by all animals, alongside humans, and can be triggered by many forms of stimuli in the environment, as observed in Goldstein’s study where he observed adrenaline responses to stress (Goldstein, 2011). Humans, scientifically known as Homo sapiens, also value fight or flight responses in the sympathetic nervous system in order to ensure survival from underlying dangerous situations. An observational study was conducted to evaluate whether or not fox squirrels and humans would undergo similar fight or flight responses after hearing an audible predatory stimulus. As observed from the book Wildlife and Recreationists part Ⅱ written by Geri W. Gabrielsen and E. Norbert Smith, when an animal gets pursued by a predator, it must to strive in order to out-run the predator. On the other hand, if an animal gets cornered by a predator, it has to go on the defense to try to fight off its aggressor (Gabrielsen, 2013). For humans, as stated by Maarten H. Jacobs, emotional dispositions towards wildlife can correspond to personal experiences and culture which can influence an individual's emotions towards different species of animals (Jacobs, 2012). From these sentiments, we hypothesized that when fox squirrels are exposed to an audible stimulus, they are likely to acknowledge the sound produced by the speakers as a threat and quickly escape away from the speaker. We also hypothesize that humans will tend to take a step away from the direction of the speaker inducing the sound since it would be perceived as a threat.

Fox squirrels are known to be agile species and usually tend to escape from human interaction, certainly because the squirrels tend to see humans as threats. Squirrels, humans, and countless other animals observe a similar trend in either escaping a given stimulus or standing one’s ground. As stated in the book, The Wisdom of the Body by Walter B Cannon, the fight or flight response is a physiological response induced by a harmful or threatening stimulus (Walter, 1967). Animals react to the threatening stimulus by their sympathetic nervous system in which it stimulates the body’s fight or flight responses given the certain stimulus (Brodal, 2004). One of the genes primarily responsible for the sympathetic nervous system’s fight or flight response is called the ADRB2 gene (adrenoceptor beta 2) (Emrick et al, 2010). ADRB2 is located on the long (q) arm of chromosome 5 at position 32 within a human’s DNA. ADRB2 associates with a receptor (beta-2-adrenergic receptor) which allows for a chain reaction to occur in order for the gene to complete its function. (National Center for Biotechnology Information, 2019). Understanding and observing the fight or flight responses within humans may be crucial to determine the similarities that reside within the fox squirrels’ fight or flight responses, which can correlate to gene similarities with humans and fox squirrels. In order to observe the presence of the ADRB2 gene within both humans and fox squirrels, a PCR (Polymerase Chain Reaction) test is required. PCR is a technique used to make multiple copies of a segment of DNA of interest or produce a large number of copies. Specifically, PCR allows the production of millions of copies of a specific DNA sequence from an initially small sample or sometimes can even be a single copy (Gilpin, B., 2007).

In our study, we focused on observing the behavior of fox squirrels, Sciurus niger, and humans, Homo sapiens, while using playback vocalizations of auditory predator stimuli. Animals tend to acknowledge predatory stimuli that a predator yields which would allow for animals to react accordingly in order to benefit their own wellbeing (Magrath, 2014). Fox squirrels need to weigh the risks and benefits of forging while being in constant potential danger (Wassmer, 2016). This research was aimed to observe how fox squirrels reacted to different vocalizations and compare it to how humans reacted. A portable speaker was used, alongside a control of not playing any vocalizations ten meters away from squirrels along the River Trail behind Holmes Hall at Michigan State University in East Lansing, Michigan. The River Trail was chosen to conduct our study due to the squirrels being most abundant in that area. The method of using playbacks to provoke a fight or flight response in both fox squirrels and humans will help to observe these behaviors and compare them to each other.

In addition to this experimental test, we conducted an observational study on the natural behavior of wild Fox Squirrels compared to humans on the Michigan State University College campus. We have observed the two species, we predicted that squirrels would show low levels of alarm responses, given that they are more habituated to human presence, just as Robert McCleery found in a study of fox squirrels’ undisturbed behavior (McCleery et al, 2007). This collected data on natural squirrel behavior and human behavior in this environment was focused on behavior in the absence of specific playback sounds. We expanded on the study of alarm behavior in urban animals from Sarah Partan and her colleagues, by providing data on signaling behavior in the presence and absence of conspecifics, which are, in this case, the surrounding squirrels and surrounding human beings (Partan et al, 2009). Including data without any outside interference, which in this case would the predatory playback vocalizations, serves as a negative control. This is because we expect no significant response from the fox squirrels or humans when simply recording their natural behavior, so it serves as an accurate medium of comparison to determine how significant the results of the playback experiment were. Coupling this control test with the experimental test previously explained would allow us to appropriately determine how much auditory predator stimuli influence the fight or flight response, or lack thereof, in both fox squirrels and humans.

Squirrel Observations

Fox squirrels commonly forage and roam around in areas surrounding forestry, in both open and secluded areas. In natural habitats like these, squirrels are likely to perceive auditory predator stimuli as a threat and their sympathetic nervous system would be subject to activation. Therefore, the studies were conducted on Michigan State University's campus along River Trail starting from behind Holmes Hall, and stretching all the way to behind Wells Hall. In order to observe how squirrels react to predatory stimuli compared to when no stimulus was present, a Sony SRS XB30 speaker was set up ten meters away from River Trail. Connected to the speaker, was an iPhone X administering a canine (German Shepherd) barking sound for two minutes and a hawk alarm call for the same duration of time. Data was observed and recorded on a OnePlus 7 Pro smartphone. Recording trials is reliable for capturing data that could have been missed when first observing the trials in the field. After administering the canine bark for five trials, the hawk alarm call played for two minutes while we observed and recorded the reactions to that vocalization for another five trials.

Human Observations

In this portion of our experimental study, we tested how humans responded to the same stimuli utilized before: a hawk screech and a German Shepherd bark. Figure 3 displays the data from this test. The most prevalent response was the subtle visual cue (46%), as many humans heard, but likely did not feel threatened by the sounds of the German Shepherd or hawk. Furthermore, flight (28%) was more common than fight (10%) for both predators, as the human subjects did not feel the need to defend themselves against two smaller animals; the sympathetic nervous system, when triggered, valued distancing the subjects from the threat more than defending against it. Lastly, the lack of responses (16%) did not dominate the data, but it was present for both, as quite a few students walking around River Trail will have their focus placed elsewhere, both in terms of their minds and their auditory senses. The conducted ANOVA test for this experiment resulted in a p-value of .0258, which is less than .05. This proves statistical significance for the hawk screech with the differences between fight, flight, and subtle visual cue for the hawk screech, and the difference between flight and subtle visual cue for the german shepherd bark. All in all, we were able to conclude from these results that subtle visual cue was the most common response for humans and that flight is a more prevalent reaction than fight for humans. We also determined that the fight or flight response is more easily triggered in fox squirrels than in humans by comparing the frequencies of responses from Figure 1 and Figure 2.

Controls

A negative control towards both humans and fox squirrels was needed to observe behaviors of both species during a period where no playback calls were being produced. This data is needed to compare the results from behaviors that have reacted to the acoustic predatory stimulus from the previous methods. Therefore, we observed the two unbothered species naturally without expecting activation of the sympathetic nervous system. Firstly, humans were observed from a staircase of a back door of McDonnel Hall approximately 20 meters away from the part of River Trail that stretches along from behind Holmes Hall towards the crosswalk of Bogue Street. Fifty humans were observed and their actions were recorded on a notebook, which consisted of categories of looking straight, looking down at phone, depending if they were walking or biking. The second control study was observing fox squirrels during no playback calls being active on different locations through the River Trail, from behind Holmes towards Wells Hall, depending on the presence of the squirrels. The squirrels were observed and recorded using the OnePlus 7 Pro and their behaviors were recorded. The behaviors noted for a total of 50 squirrels includes foraging, pouncing, tail flicking, or eating.

Primers and PCR/Gel Electrophoresis

After obtaining data on the behavior of both species, a Polymerase Chain Reaction (PCR) test was required to investigate the presence of the ADRB2 gene within humans and amplify the corresponding genes. Two separate primers for the ADRB2 gene, were used for gene amplification. Primer 1 had the sequence: 5’-CAAGTACCAGAGCCTGCTGACC-’3, and Primer 2 had the sequence: 5’-TGAAGAAGGGCAGCCAGC-3’, which were both found using similar methods to the research done by B.J. Jungerius and his colleagues (Jungerius, et al 2003). To proceed with PCR, human DNA was obtained from the salivary glands of four human subjects. Once the DNA was extracted, PCR was used to amplify the desired genes. The PCR steps were acquired from Jungerius’ paper as well. The master mix containing the human DNA was carried out in 50 µl increments. Each mix contained 11 µl of extracted human DNA, 1 µl dNTP Mixture, 5 µl 10 x PCR buffer, 1 µl of the forward and backward primers, 4 µl of blue dye, 30.5 µl of DI water, and 0.5 unit HotStar Taq DNA Polymerase, which enables the DNA replication at high temperatures. The mixtures were kept in ice until usage. The mixtures were then put into a thermocycler. First mixtures were denatured at 95 °C for 5 minutes, followed by 25 cycles at 95 °C for 30 seconds each, annealing for 45 seconds at 62 °C. Elongation proceeded at 72 °C for 3 minutes, and the final elongation was at 72 °C for 10 minutes. The amplified DNA was then used for conformation through gel electrophoresis. The Edvotek guide to forming the agarose gel was utilized. To make the gel, 5 mL of a 10x Lithium Borate was added along with 0.50 grams of agarose which was diluted with deionized water for a total of 50mL. The solution was then placed in a microwave on high for 1 minute, maintaining that it did not boil, and it finally was mixed so that the agarose could completely dissolve. The agarose solution was then cooled to 60 °C while resting, and 0.5 µl of red dye was added to it. The cooled agarose solution mixed with dye was then poured into the gel casting tray and left to solidify for 30 minutes. After the gel solidified the samples were placed into the wells. Well 1 contained 5 µl of Gold Bio’s 100 base pair ladder, Well 2 contained 11 µl of the ADRB2 gene target DNA which was to be amplified by PCR along with 4µl of blue dye.

Data Analysis

An ANOVA Test was used in order to test for the significance of the differences in frequency between the various responses for both the fox squirrel study and the human study. The same was done for the human experimental study. A null hypothesis was created in order to run this statistical test. In this ANOVA Test, the null hypothesis stated equal frequencies are to occur for fight, flight, subtle visual cue, and no response. If the null hypothesis was proven true then there would be no statistical significance between that data collected and it would fail to reject the null hypothesis. The alternative hypothesis would state that the frequency for fight, flight, subtle visual cue, and no response were significantly different. The test has resulted in a p-value which is used to determine statistical significance between the variables. If the p-value was below 0.05 then the results were considered significant.

Fox Squirrels' Results

Fox squirrels reside in an environment where the predation risk is quite low compared to squirrels in urban areas. Alternatively, fox squirrels are still prey to hawks (ariel predator) and canines (terrestrial predator) in the general vicinity. To illustrate, the squirrels stopped their foraging once they acknowledged both the hawk and canine vocalizations and reacted accordingly. For the aerial predator, in our sample size of twenty-five squirrels, a flight response was observed 92 percent of the time as depicted in Figure 1. The squirrels stopped foraging and reported to a low area while remaining alert. This is because the squirrels want to remain hidden from the hawk and the closer to the ground they are, the more difficult it is for the hawk to hunt them down (Gibbens, 2017). With the terrestrial predator, a flight response was observed 72 percent of the time, as shown in Figure 1. The squirrels stopped foraging and climbed a tree. This was most likely because German shepherds cannot climb trees, and the squirrels felt safe retreating to higher grounds (Gibbens, 2017). We observed that 12 percent of the time, squirrels showed a fight response to the hawk vocalizations and 8 percent of the time to German shepherd vocalizations. This is because fox squirrels are more likely to attempt to flee a predator rather than to try to fight one off (Gabrielsen, Geri W., 2013). Twenty percent of the time, there was no response during the whole study. To analyze the statistical significance of these results, we ran an ANOVA Test and found that the results were statistically significant.

Humans' Results

In this portion of our experimental study, we will test how humans respond to the same stimuli utilized before: a hawk screech and a German Shepherd bark. Figure 3 displays the (predictive) data from this test. In this context, fight represents anytime human subjects acknowledge the sound being produced, and slow down, implying the intention of protecting themselves. In contrast, flight represents anytime they acknowledge and proceed to move faster, implying an intention to escape. Subtle visual cue refers to anytime the humans acknowledge the sound, but don’t alter their movement in any way (Ex: Person turns his/her head toward the sound, but continues walking by at a normal pace). Finally, it will also be noted whenever subjects exhibited no response physically whatsoever. We predict that the most prevalent response will be the subtle visual cue, as many humans will hear, but may not feel threatened by the sounds of a German Shepherd or a hawk. Furthermore, we predict that flight will be more common for the German Shepherd bark, and fight will be more common for the hawk call, due to the perceived size difference and intimidation factor of the two different animals. Humans will likely feel safest defending themselves against a hawk, as opposed to a German Shepherd, which they may want to distance themselves from. Lastly, the lack of responses will not dominate the data, but it will be present for both, as quite a few students walking around River Trail will have their focus placed elsewhere, both in terms of their minds and their auditory senses. All in all, we predict that the data we collect from this experiment will allow us to conclude that the fight or flight response is more easily triggered for squirrels than it is for humans.

Control Results

Both humans and squirrels naturally do not react when given no stimulus. Figure 3 indicates that the observations show that a fight or flight response is not commonly exhibited by the two studied individuals. Without the presence of something that is terrifying, either mentally or physically, the acute stress response that results from the activation from the sympathetic nervous system does not stimulate these responses. Observations for humans included walking looking forward (30%), walking looking down at their phones (36%), biking looking forward (26%), and biking looking down at phones (8%). Observations for squirrels included foraging (10%), pouncing (36%), tail flicking (32%), or eating (22%). In addition to this control, random sounds produced by the surroundings in the outdoors at River Trail could have affected these observations. Our recordings contained other noises that potentially could influence either of the two species' behavior, which is similar to what Lilly MV, et al. recorded in their study on grey squirrels and bird chatter. These additional sounds caused squirrels to retreat to lower grounds, and humans to glance up from their phones and look around. Also, when squirrels interacted with each other, the dominating squirrel scared off the other squirrel it was in contact with, resulting squirrel to retreat far away. These natural observations show a potential fight or flight response, but do not greatly affect our data.

PCR and Gel Electrophoresis Results

The results of the gel electrophoresis shows the presence of the ADRB2 gene found within the amplified ADRB2 human DNA extraction. Figure 4A depicts the results from the gel electrophoresis with Well 1 having the Gold Bio 100 base pair ladder, and Well 2 having the apmified ADRB2 gene. The smear located at and above the 455 base pair line might show evidence of the ADRB2 gene. Even though we have predicted a single line similar to the ladder the gel electrophoresis appears to show two different results. This may have been due to an error while conducting the PCR master mix. The smear below and above the 455 base pairs maybe due to the degradation of the nucleotide strands which disallows the single line to show.

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Figure 1: Number of responses of fox squirrels to different types of vocalizations. Illustrates results for fox squirrel responses (n=50) to hawk vocalizations and German shepherd aggressive barking. Playback vocalizations of the hawk and German shepherd aggressive calls were played on a speaker (Sony SRS XB30) and was conducted on Michigan State University's campus along River Trail starting from behind Holmes Hall towards Wells Hall. A fight response was categorized as a squirrel flicking its tail and approaching the speaker. A flight response was categorized as a squirrel fleeing the scene of where the speaker was playing the vocalization. No response was categorized as a squirrel having no reaction towards the speaker. Error bars were made by finding the standard deviation through an ANOVA test on a graphic calculator. Error bars represent standard error between each sample. Error bars not overlapping represent statistical significance plus or minus 5%. ANOVA test analysis ran on data points and p-value given was 0.012 which is less than 0.05 proving statistical significance.

Figure 2: Experimental Results of Human Responses to Predator Stimuli: Illustrates four different types of behavioral human responses to squirrel predatorial stimuli. Playback vocalizations of the hawk and German shepherd aggressive calls were played on a speaker (Sony SRS XB30) and was conducted on Michigan State University's campus along River Trail starting from behind Holmes Hall towards Wells Hall. Five experimental trials of 5 human samples for each predatorial vocalization, for a total of 25 human responses per predator. In this context, fight represents anytime human subjects acknowledge the sound being produced, and slow down, implying the intention of protecting themselves. In contrast, flight represents anytime they acknowledge and proceed to move faster, implying an intention to escape. Subtle visual cue refers to anytime the humans acknowledged the sound, but did not alter their movement in any way (Ex: Person turns his/her head toward the sound, but continues walking by at a normal pace). It was also noted whenever subjects exhibited no response. Error bars were made by finding the standard deviation through an ANOVA test on a TI-84 graphing calculator. Error bars not overlapping represent statistical significance of plus or minus 5%. ANOVA test analysis ran on data points and p-value given was .0258 for flight, fight, and subtle visual cue under the hawk screech and only flight and subtle visual cue for German Shepherd bark, which is less than 0.05, thus proving statistical significance. Areas on the graph with error bars overlapping does not show statistical significance.

Figure 3 Controls: Behavioral responses in humans and fox squirrels (control). A Number of observational responses in humans (n=50). Observations included walking looking forward, (30%) walking looking down at phone (36%), biking looking forward (26%), and biking looking down at phone for humans (8%). B Number of observational responses in squirrels (n=50). Observations included foraging (10%), pouncing (36%), tail flicking (32%), and eating for fox squirrels (22%). In addition to this control, random sounds produced by the surroundings at River Trail could have affected these observations. Our recordings contained other noises that potentially could influence either of the two species' behavior, which is similar to what Lilly MV, et al. recorded in their study on grey squirrels and bird chatter. (Lilly MV, et al. 2019). There is no standard error due to the fact that the sources of error did not influence the data we quantified.

Figure 4: Gel Electrophoresis of Human ADRB2 Gene: Figure A shows a picture of the gel electrophoresis results with Well 1 containing a 100 base pair ladder which labels the base pairs descending from 1,000 to 100. Well 2 contains the ADRB2 gene amplified by PCR which was calculated to have an estimate of 455 base pairs. Under the 455 base pair result another result was found with less than 100 base pairs. Figure B shows a graph indicating the gel results with the y-axis showing the molecular size in base pairs and the x-axis showing the migration distance in cm from the wells. The blue dots indicate the rungs of the 100 base pair ladder. A best line of fit was also made with an associated R squared value of 0.91201 which indicates a relative regression model. The Red line on the graph indicates that the ADRB2 gene is 455 base pairs long and has migrated 2.19 cm.