Chromosome one of the human genome contains an important gene that is called STMN1, which controls both learned and innate fear expression. The purpose of this project was to examine how the STMN1 contributes to varying fear expression of both squirrels and humans in rural and urban environments. It was hypothesized that both humans and squirrels in a rural environments will experience increased sensitivity to playbacks in the experiment when compared to their urban counterparts because they have adapted to their noisy environment due to the expression of the STMN1 gene controlling learned fear. (Fisher,2009) Observational playback experiments were conducted in order to document and quantify reaction behaviors humans and squirrels displayed in both environments. Additionally, polymerase chain reaction was conducted in a 2% agarose gel with human DNA to verify the presence of the STMN1 gene at 270 base pairs (Zhang,2016). This research is significant in science because it works toward identifying what genes humans and animals might share that have been passed down by a common ancestor and how that has affected the survival of a species over time. It was predicted that organisms that have been repeatedly exposed to human stimuli reduce their anti-predator response (McCleery, 2009). The results showed that subjects in urban environment had a lower score distribution and in general reacted less overall when compared to subjects found in rural environments, that often reacted drastically to fear stimuli produced by the playback.

         Urbanization across the country has created new environments for many different species of wildlife. Many species have had to alter their population dynamics, physiology, movements, habitat selection, and behavior in response to new surroundings (McCleery, 2009). Fox squirrels (Sciurus Niger) use many different techniques to communicate to one another. Specifically, fox squirrels use different physical and auditory cues when reacting to predators in order to communicate that there is a threat to other squirrels in the area (Rosier & Langkilde, 2011). When a squirrel hears a potentially life threatening noise, they often react to it with a threat response. These responses could include freezing in place and looking around while they are quadrupedal, or on all fours, bipedal, hind legs, looking around and listening, and a flight response, which is fleeing the area. However, it has been hypothesized by McCleery that organisms that have been repeatedly exposed to playback stimuli reduce their anti-predator reactions (McCleery, 2009).

          In all animals, stressors cause instinctual vigilance, which is the mental capacity to sustain attention over extended periods of time, as a safety precaution to possible threats (Unck, 2009). These can be found in both squirrels and humans as natural responses to stressful situations. As expansive as humans are, there are many animals, such as squirrels, who can be found in both urban settings with humans and rural, isolated areas without them. It is known that animals that are surrounded by humans throughout their lifetime will modify their behavior to fit the environment (Dishkoff, 2006). How much does this change instinctive reactions to stressful situations, and is it possible that squirrels, in particular, are able to adapt to danger in new forms? The same can also be asked of humans, if the level of vigilance to a threat is different in different settings, as well as if some threat would elicit more of a response in one setting rather than another. Therefore, the group tested if the location of a stressor has an effect on the degree of the response.

          Though they are different species, when compared to squirrels, humans do exhibit some homologous, or similar, behaviors. In the case of homologous threat response behaviors between squirrels and humans, the STMN1 gene , may be responsible for how they react to possible threatening sounds. The STMN1 gene, or stathmin gene, has been linked to control both learned and innate fear; the gene is located in the amygdala, which is a part of the temporal lobe of the brain (Gleb, 2005). As such it is easy to infer that this gene may be divulged to both species from a common ancestor, and as a result has ensured the survival of both species as time has gone on. Additionally, it can be predicted that the STMN1 gene is responsible for how both humans and squirrels react to playback stimuli.

          With the sounds and location chosen, it is predicted that there will be a clear distinction between the level of reactions of the squirrels and the reactions of humans to sounds in different locations, urban and rural. Vigilance is important and is key to survival (Unck, 2009). Humans are expected to react more to airhorns, squirrels will react to hawk calls, and both will be alert with dog barking sounds. This is because both organisms share the STMN1 gene that allows both squirrels and humans to be more alert when they sense a possible danger or threat (Fisher, 2009; Gleb, 2005). The group expected that the degree of reactions will have differences in the different (urban and rural) locations. However, both humans and squirrels reacted in equal amounts to the dogs because it is a sign of possible danger.

        Twenty fox squirrels (Sciurus Niger) of varying sex were observed to see how they reacted to different types of auditory stimuli and how the reactions differed between urban and rural areas. On Michigan State University's campus, the red fox squirrels were observed in the area behind Wells Hall and along the River Trail, which had a multitude of trees and smaller grassy and open areas, and the Botanical Gardens, which had trees, bushes and low lying plants, and grassy open areas. These locations were picked because of the varying levels of foot traffic in each location; the area behind Wells Hall has the most amount of foot traffic, as the River Trail is used by students to get to classes and Wells and the surrounding Halls. In this experiment, foot traffic was defined by the amount of students, professors, and other people who walk through each area on a day to day basis. These squirrels were observed on grassy areas with lots of canopy around because according to research done by Robert McCleery, squirrels prefer these areas when they are active (McCleery, 2010). These experiments were performed on squirrels who did not seem to be disturbed by the group or other animals and that were not interacting with other squirrels (Greene, 1998). A playback experiment was used in order to determine the levels of reaction of the fox squirrels to the sound of a red tailed hawk (Accipiter cooperii) call, a gas air horn, dog barking, and bird chatter. A red tailed hawk call was used as auditory stimuli because it is a natural predator of the red fox squirrel (Thorson, 1998). Bird chatter was used as a call in order to have a negative control. Recordings of the red tailed hawk call, the semi horn, and the bird chatter, that were obtained from YouTube, were played on an iPhone X or an iPhone 7 and relayed through a Bose SoundLink Mobile speaker or a Fugoo speaker once there was a squirrel that was approximately 10-15 feet away from the speaker. No bait was used. Observations were recorded of the squirrel one minute prior to the call. The call was played 10 seconds, and the reactions of the squirrel were recorded 3 minutes after the call was played (Leger, 1978). Observers were located approximately 10 feet from the speaker in order to see the reactions. These same procedures were repeated at each of the locations.

        Similarly to testing squirrels, humans were tested in different locations of more “urban" against more “rural" to observe responses. Observations were made on random humans at Michigan State University to see their responses to different potentially threatening playback noises in the different locations. These tests were done in both urban and rural settings located at the Cata Bus Station and the River Trail behind McDonnel Hall. These locations were used because of the varying levels of foot traffic, McDonnel Hall having the least and the Cata Bus Station having the most. The sounds used for the playback experiment were bird chatter, a dog barking, airhorn, and hawk sound. For audio of a threatening dog, Pongracz used recordings of Schutzhund, protection dogs, which have training that encourages aggressive barking (Pongracz, 2006), so audio of a similar barking style was selected. All playback sounds can be found on YouTube. At the location a small bluetooth speaker was set up in an area near where people would naturally be or be passing by. (Leger, 1978) No bait was used. Observers conducted the observations at approximately 10 feet away from the speaker. From there, the selected sounds were played from an iPhone connected to a speaker and observers recorded what level human subjects individually reacted to them. The trials and responses were also recorded on an iPhone camera. Observations were made from one minute before and three minutes after the sound was played, during which the observer recorded the behavior (Leger, 1978).

        Leger and Owings used a score system out of four to indicate the average position of a squirrel over one trial, so we decided to also use a scoring system out of four, but altered slightly. Horizontal position, where all 4 of the feet of the squirrel are on the ground and the head is at the same level or lower in relation to the hind legs (Leger & Owings, 1978), received 1 point. Elevated position, where the squirrel's head is raised in relation to its hind legs and one or both of the forelegs are raised (Leger & Owings, 1978), received 2 points. Vertical position, where the squirrel is standing upright with hind legs fully extended (Leger & Owings, 1978), received 3 points. Any form of locomotion, such as running away or climbing up a tree, received 4 points. Then, while watching the videos we recorded or squirrels and humans, we noted any change in posture and recorded it, then found the mean score in order to give the average posture score over the entirety of the recording. According to research done by Blanchard, both male and female humans investigate when there is an unexpected noise, and run away when they are in direct danger, so we did not differentiate between males and females (Blanchard et al., 2001). For humans, a similar scoring system as the squirrels was used. A four-point score keeping system was used to quantify the data. 1 point when the subject wasn't impacted by the sounds and their position and posture did not change. 2 points if they moved their head around in a swivel or side to side motion without changing their position. 3 points if they are actively searched for the sound. 4 points if they consciously removed themselves from the situation. To analyze the data collected through observation, an ANOVA statistical test was conducted to compare the scores between urban and rural locations, for both humans and squirrels respectively, with statistical significance found with p<0.05.

        Polymerase Chain Reaction was used to make copies of DNA fragments in order to amplify the the STMN1 gene in the fox squirrel and human genome. In order to perform PCR DNA, obtained from cheek cells from the processed saliva of our group members, were used to amplify the target segments. To perform PCR we make a 50 µL solution consisting of 1µL of Taq polymerase, 1 µL of each of the primers of 5'-AAGAAAGACGCAAGTCCCATG-3' and 3'-ACCCTGCTGACGAGACTGAAG -5' (Zhang, 2016)(forward and backward respectively), 1 µL of DNTP providing free nucleotides, 10 µL of our DNA, 5 µL of our PCR buffer (10x), 2 µL of MgCl2, with the rest of the volume diluted with deionized water (~30 µL). The mixture was put into a thermocycler with 30 seconds of denaturation at 95 ℃, 30 seconds of annealing at 58 ℃, and 30 seconds of polymerizing at 72 ℃, cycling for ~90 minutes (Zhang, 2016). The PCR was loaded into the wells of a 2% agarose gel in a mixture of 10 µL of PCR solution and 5 µL of dye. The agarose concentration was increased to 2% because of the short length of our target DNA, being only 270 BP long (Zhang, 2016). Gel electrophoresis was run at 200V for 15 minutes with a 100 BP ladder along side the PCR to compare length of target DNA.

        Squirrels and humans had different levels of reactions when exposed to fear stimuli. On average, 59.75% of human reactions received a score of 1 (n=82), whereas only 35.480% of squirrel reactions received a score of 1 (n=31). 41.93% of squirrel reaction received the highest score of 4, but 0% of humans received this score. Looking at the trends, majority of humans received low scores based on their reaction, meaning that they did not react as extremely to the fear stimuli compared to the squirrels. Majority of the squirrels received high scores, meaning that they generally had more of an extreme reaction in response to the fear stimuli (Figure 1).

        Our experiment began with administering bird chatter, the negative control to squirrels from both areas. As seen in figure 2, the squirrels from the rural areas (n=4) scored 1.75 points on average as opposed to the urban squirrels (n=8) who, overall, scored 0.75 points less. We then proceeded to use the sound of a dog bark in which both the urban (n=5) and rural squirrels (n=3) scored an average of 4 on the scale. Using the hawk screech, the rural squirrels (n=4) scored an overall 4 as well; However, the urban squirrels (n=5) only scored an average of 2.75. As the data in figure 2 indicates, the test utilizing the airhorn yielded an average score of 3.67 for the rural squirrels (n=3); the score for the urban squirrels (n=5) was an average score of 3.2. In general, the squirrels exposed to the dog bark reacted more severely whereas squirrels exposed to the bird chatter reacted less severe, as previously predicted. As for the airhorn and red tailed hawk call playbacks, that were not controls, it was found that, on average, the squirrels reacted in a similar trend: with rural squirrels reacting more drastically, and urban squirrels reacting less drastically when exposed to these two playbacks. However, all possible p-values calculated were greater than 0.05, indicating that there is no statistical significance. Meaning the null hypothesis, squirrels will have the same level of fear response, regardless of location, was accepted.

        When looking at the reactions of humans in response to playback stimuli. Overall there seems to be more instances of a greater degree of reaction to specific sounds when played in the rural environments as opposed to urban environments. The reactions of the humans were ranked on a scale from one to four with one representing no discernable reaction and four representing the highest level of reaction. Responses in general for humans did not rank very high on our scale, generally staying within a one and two, with person reacting at a level of three (figure 3). The average responses to playback sounds are more severe in rural areas as opposed to urban areas and that the difference between the two locations show statistical significance, finding a p-value of 0.0078 (figure 3). This data supports the notion that humans have different levels of fear, learned depending on the environment. When comparing urban and rural environments, the same sounds in the urban settings have elicited a lower overall response in humans than in their rural counterparts.

        Using Polymerase Chain Reaction, it was tested whether both Red Fox Squirrels (sciurus niger) and Humans (homo sapiens) share the STMN1 gene as it would explain similar reaction behaviors in response to playbacks. The STMN1 gene has 270 base pairs which, as shown in Well A in Figure 4 is where it generated a marker in the 2% agarose gel. This was corroborated by the 100 bp ladder located in Well F. (Figure 4) Based on these results, it was concluded that, since human DNA was tested, humans have the STMN1 gene, which controls both learned and innate fear (Fisher 2009), which affects how subjects react to playback stimuli if they are continuously exposed to it. This supports the hypothesis that Fox Squirrels genome also contains the STMN1 gene, however, since it was not tested using Polymerase Chain Reaction it cannot be concluded for certain.

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Figure 1: Reaction Score Percentages of Squirrels and Humans. A. This figure shows the percentage of squirrels and humans that were given each score based on their reaction to different stimuli. The scoring system was ranked from 1 to 4. The scales were slightly different for squirrels and humans. For squirrels, a score of 1 means that all 4 of the feet of the squirrel are on the ground and the head is at the same level or lower in relation to the hind legs. A score of 2 means the squirrel's head is raised in relation to its hind legs and one or both of the forelegs are raised. A score of 3 means the squirrel is standing upright with hind legs fully extended. A score of 4 means any form of locomotion, such as running away or climbing up a tree. For humans, a score of one means that there was no discernible reaction or change in behavior by the person we were observing. A score of 2 means that the person looked around in response to the playback. A score of 3 means that there was a more active search and change of behavior to the sound. A score of 4 means that there was a conscious effort made by the person observed to remove themselves from the situation in response to the sound. An ANOVA test was conducted and the p-value indicated that the data was not statistically significant. B. This figure includes all of the different sounds that were using for the playback experiment, for both human and squirrels, including bird chatter, dog barking, a red tailed hawk call, and an airhorn.

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Figure 2: Compared average response level of Fox Squirrels (Sciurus niger) in urban and rural environments. A.This figure shows the average response level to certain auditory stimuli within different locations. The squirrels' responses were based on a four point scoring system, which came from Leger and Owings' 1978 paper, "Responses to alarm calls by California ground squirrels: Effects of call structure and maternal status" (Leger & Owings, 1978). The scores were based on the highest response level that the squirrel showed in the duration of each video. A higher score would be indicative of a squirrel with more reactive behaviors when the sound was administered. Both bird chatter and dog barks were used a controls, but bird chatter was used as a negative control while dog barks were used as a positive control. The controls were used in the context of how humans would tend to react in comparison to the squirrels to these sounds. The sample size used for the urban area (n= 23) was vastly larger for the urban area than that of the rural area (n=14). An ANOVA test was run between the rural and urban squirrel datasets, as well as between individual playbacks in both areas. For the overall test, the p-value was 0.115, which means the data was found to be not statistically significant. The bird chatter (p= .167), hawk screech (p=.089), and airhorn (p=.432) were all not statistically significant. Dog barks elicited the same reaction in all trials, so an ANOVA test could not be performed. Standard error bars were calculated as one standard error per region. B. This shows a squirrel displaying a level 3 response to one of the playbacks.

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Figure 3: Reactions levels of humans to playbacks in urban and rural locations: A. The figure shows the results of the experiment testing levels of reactions of humans to playbacks in both urban and rural environments. Responses to the sounds were quantified on a scale of one to four, as adapted from Leger and Owings scale used for quantifying observed behaviors (Leger & Owings, 1978). Higher scores represent a more severe response by the subject in response to the playback. The urban location for the observations of humans was by the CATA bus station on campus. For the rural location, observations were made on the trail behind Mcdonel hall. Four different playback sounds were tested, of bird chatter and dog barking, serving as positive and negative controls respectively, as well as playbacks of a red tailed hawk and an airhorn. Error bars on the graph were calculated to one standard error. An Anova test was done comparing the responses of humans between urban (n=34) and rural (n=48) locations to find a P-value of 0.0078, supporting that there is significance in the difference of location (for p<0.05). B. This is an example of human reaction that would be classified as a 2 on the scale.

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Figure 4: Polymerase Chain Reaction (PCR) and How it is Used to Test For the STMN1 Gene in Humans and Fox Squirrels. A.This is a figure of what the gel electrophoresis of the STMN1 gene in humans looks like. Since the gene was only 270 base pairs, the agarose concentration in the gel was increased from 1% to 2% in order to capture the gene during electrophoresis.The forward PCR primer used for the STMN1 is 5'-AAGAAAGACGCAAGTCCCATG-3'(Zhang, 2016) and the reverse primer used was 3'-ACCCTGCTGACGAGACTGAAG -5'(Zhang, 2016). Well A contained the human DNA sample and Well F contained the 100 bp ladder that was used to compare how many base pairs appeared after leaving well A. B Is a graph composed of identified base pairs from the molecular ladder and their migration distance from the well. Migration distance from well was measured using an online pixel ruler and then converted to centimeters using an online centimeter to pixel converter. The graph was analyzed using a logarithmic scale and an exponential line of best fit was generated in excel using the data along with an R2 value, which measures how close the data is to the fitted line of regression. Additionally, the band that appeared in Well A migration distance was measured using an online pixel ruler and then converted to centimeters to determine where it fell in relation to the DNA ladder.