Urbanization is the process in which a city acquires more residents, thus increasing the population density (McCleery, 2009). However, when a city becomes urban, humans are not the only organisms that adapt to city life, animals must adapt as well. Amongst the prevalent animals in the city, a prevalent species to Michigan State's campus in East Lansing, Michigan is the Eastern Fox Squirrel. A behavioral difference of urban squirrels compared to rural squirrels have been frequently observed, this behavior is correlated to the accustomization of living in such close proximity to humans and adapting to humans as a stimuli (McCleery, 2009). A predominant reason for having their natural anti-predator behavior reduced is natural selection. The benefits outweigh the costs of having anti-predator behaviors (Blumstein, 2002). With such behaviors diminished the Fox squirrels will have more time to do activities that are important to survival, such as foraging for food (Blumstein and Daniel, 2005).

The Eastern Fox Squirrel is an exceptionally common mammal in Michigan. This species of squirrel is diurnal and reside in areas ranging from rural to urban domains (McCleery, 2009). Their food resources include a variety of hard shell nuts, which depend on abundance and digestibility; walnuts, acorns, and hickory nuts are their main consumption (Smith and Follmer, 1972).They also have a defense mechanism that can help protect them against predators. Defense mechanisms can entail alarm calls, and motor behaviors such as running or freezing. This innate behavior is called, anti-predator behavior. When defense mechanisms are being used, the squirrel stops its activity at hand and becomes alert by freezing, listening, and scanning the area (McCleery, 2009). It is similar to a human's fight-or-flight instinct. However, as previously stated, squirrels who live in an urban environment have scant anti-predator behavior, it is believed that this is a result of the habituation of the species (Parker et al., 2008). The behavior we are specifically looking at is the difference in reactions to threats based on the environment. We believe that humans and squirrels will have similar behavior patterns based on similar genome structure, twelve common chromosomes (Stanyon et. al, 2002). For example, we believe that humans living in a rural setting may be more on edge, attentive, and responsive to threatening stimuli. This may be due to the low amount of stimuli in rural environments compared to an urban environment. Interestingly enough, even seemingly little interference can completely change the responses of individuals to stimuli (McDonnell and Pickett, 1990). In connection to this, humans and squirrels both have a fight or flight response which describes the way in which an individual response to stressful situations. It's believed that vigilance directly corresponds with compromise between risk and energy (Frid and Dill, 2002). Studies have shown that environments have an influence on the modification of mammal's DNA (Provencal, 2011). For example, humans in rural area are not as exposed to a constant source of stressful stimuli, this results in, humans living in a rural environment appearing to demonstrate a greater fear response to stressful stimuli than those humans living in an urban setting that are consistently exposed. This phenomenon could be explained by a concept known as epigenetics (Provencal, 2011). Epigenetics, the study of a change in phenotype without altering the genotype, has shown that although genes will not be altered they can be modified from environmental influences. Stressful or high stimuli environments have shown a modification in the epigenome in which a mark has been left on a specific gene. Researchers have found that this mark influences proteins that produce hormones. The hormones released are responsible for specific stress responses and social behavior (Provencal, 2011).

Vulnerability of squirrels can be concerning as this diminishes anti-predator instinct, this could affect the environment around us and allow the evolution of the commonly known Eastern Fox squirrels (Boonstra et al., 2007. Blumstein and Daniel, 2005. McCleery, 2009) . Evolution through natural selection shows the adaptation of a species to increase survival rates (Boonstra et al., 2007). When food resources become scarce, natural selection will take over to prioritize a species foraging for survival over predatory instincts. Without the consumption of food a species cannot thrive and therefore will begin to die off due to starvation and malnutrition, to avoid this they will diminish their vigilance instinct since predators only provoke a risk of fatality (Walters and Jaunes, 1993).When threatened by a predator the natural instinct of an animal would be to "fight or flight" (McCleery, 2009). By observing their surroundings squirrels can judge the extent of a potential threat. This judgment will allow them to choose the appropriate response which may include, running, or remaining in a motionless state (McCleery, 2009). This behavior is encoded within any animal's natural response system. Is there a way this instinct could be reduced? In a squirrel's natural environment, predators such as humans are scarce and food will be of abundance, yet when faced with limited resources and constant human population, urban environments provide a challenge for foraging squirrels. This challenge has the possibility of imposing a change to the predator instincts of squirrels in order to survive. Previous studies of urban animals and rural animals have shown a distinct contrast in the natural predator instinct. Urbanized animals, such as the Eastern Fox squirrel, showed a decreased response toward traditional predators (Ward and Low, 1997. And McCleery, 2009). The constant exposure to humans in an urbanized environment have decreased the "flight" response of squirrels (Ward and Low, 1997). When the "flight" instinct is diminished this allows for squirrels to continue foraging, without the risk of losing their limited resource. Studies have found that constant exposure to stressful stimuli can physically modify DNA (Provencal, 2011). When exposed to stressful stimuli hormones are released which allow the body to appropriately react. These hormones are regulated by peptides and are found in the pituitary gland. One hormone that is released during a stress response is Adrenocorticotropic Hormone (ACTH) this release then begins the release of glucocorticoids (Boonstra et al., 2007). The gene that controls said hormone releases the Pro-opiomelanocortin (POMC), which is located on chromosome 2, or more specifically 2p23.3 (NIH, 2016). The POMC gene has been identified in both squirrels and humans.

In order to observe how habituation of Eastern Fox squirrels behavior, we plan to use playback sounds to mimic possible threats of danger, such as a car horn or a hawk call, to invoke a physical response from a sample of both urbanized and rural squirrel populations. In addition, once the playback is sounded we will time how long until a response is shown. As in a previous study conducted by researcher Kimberly Jayne, focusing on grey squirrel's response to danger signals while foraging, we want to directly compare how long it takes for a squirrel to respond to direct cues of danger and then relate that to the environment they live in (Jayne et al, 2015). We plan to apply similar methods from this previous study conducted by Jayne, such as the idea of simulating social risk by using acoustic playbacks of stimuli that the squirrels may be exposed to at a foraging patch (Jayne et al, 2015). These methods were chosen because they allow for an observational response without altering any part of the squirrel's environment. While likewise, answering our overall question by providing numerical data that may, later be analyzed to determine if there is in fact a correlation between foraging environment and a reduced response to predator cues. In expansion,the purpose of the purely observational study conducted on humans in order to determine if there is a possible link between vigilance behaviors found in both Eastern Fox squirrels and people, due to a genetic component (Stanyon et al, 2002) . The purpose of the conducted observations is to determine if the environment of a species diminishes their anti-predator instincts. We hypothesize that the urbanization of an environment will diminish anti-predator vigilance instincts to allocate more time for foraging.

Sampling Sites

Written by: Jordan Fournier

Revised by: Jacob Trowbridge

Finalized by: Jacob Trowbridge

            Our sampling sites for the squirrel experiment include two urbanized areas on Michigan State's campus, River Trail behind the International Center and the Landon/Union area in North Neighborhood. We also included two rural areas on Michigan State's campus, Baker Woodlot and the Sanford Natural Area. These locations were chosen on the basis of human presence. Our urbanized areas offer a large amount of human presences and population for the campus. The squirrels in this area have constant human interaction providing them with a more urbanized environment, while our rural locations are woodlots where there is little to no human presence. The rural areas allow for the squirrels to partake in their natural environment.

Eastern Fox Squirrel Playback

Written by: Kaitlyn Thrush

Revised by: Jacob Trowbridge

Finalized by: Jacob Trowbridge

           We began by going to one of our sampling locations and observing how long it takes a foraging squirrel to respond to a playback of predatory stimuli and also note the type of response portrayed. A JBL Charge 2+ bluetooth speaker was placed near squirrel habitat and once we were far enough away to observe, we played a downloaded call, from the internet to an iPhone 7, on full volume. We then recorded the response using a chart that we created. In addition, we recorded if no response occurred. To determine if a response occurred, we referred to, the alert postures of fox squirrels (McCloskey, 1975). This procedure was repeated in all four sampling locations, both urban and rural environments. Our predatory playback sounds included a red-tailed hawk call, a car horn, and human conversation and interaction.

Written by: Nicole Stewart

Revised by: Jacob Trowbridge

Finalized by:Jacob Trowbridge

           Our experiment was conducted four days Sunday October 16th to the 23rd, through each day we focused on one of the four specific locations for ten squirrels per sound. The JBL Charge 2+ Bluetooth speaker we used was set at full volume to make sure the squirrels could hear it. We would observe ten squirrels in one area of the location and once we had the sufficient sample number we would move to a new area of the location to play the next sound. We waited until a squirrel was within a ten foot radius and then we would play the sounds. We measured out the ten feet with a measuring tape and marked the ten feet with sticks. Once the squirrel stepped into the radius, one person played the sound while another person started a timer to get the total time the squirrel was in the radius, this person would stop timing when the squirrel left the radius. If the squirrel stopped foraging, then another person started another timer to record how long the squirrel stopped foraging and would stop that time either when the squirrel left the radius or went back to foraging. Once we had the area set up we would leave for five minutes to prevent a disturbance factor. As a way to get quality data we created a control. Our control was negative, which means that we expect no reaction from the squirrels. For our control, we placed a bluetooth speaker on the ground and walked twenty feet away in order to record a purely observational response. This was considered our control because the speaker was not playing anything. This helped reinforce our data because then we could note any reaction to the speaker itself and compare these to the reactions to those with sounds playing.

Human Observation

Written by: Nicole Stewart

Revised by: Nicole Stewart

Finalized by:Jacob Trowbridge

           The other part of our experiment focused on the reactions humans portray toward possible threats. We conducted an observational study on humans to see how they behave differently in urban and rural environments. There were 15 people we observed at each of the four locations so, we had a total of 60 human observations. We observed these people at crosswalks without traffic signals to see the number of times they look before crossing. A look was defined as one visible turn of the head towards the direction of the road, which approximately 90 degrees. We recorded this using a chart that listed how many times a person might look, one thru ten, and put a tally in the appropriate box. We also looked at whether or not the person stopped before crossing the road, which would not have a crosswalk traffic signal. Two of the rural locations we chose are the small towns St. John's and Corunna which are outside of Michigan State. The two urban locations we observed people on the crosswalks near the intersection of Wilson road and Chestnut road and the crosswalks by the Sparty statue on Kalamazoo road.

Statistical Analysis

Written by: Jacob Trowbridge

Revised by: Nicole Stewart

Finalized by: Kaitlyn Thrush

           Our experiments provided both qualitative and quantitative data. The qualitative data collected from our observational experiment was the physical response of the squirrel to the stimulus in the squirrel experiment as well as, whether or not people stop before crossing a road with no crosswalk signal in the human experiment. While, the quantitative data was the amount of time it takes for a squirrel to react to the playback sound, as well as the number of head turns exhibited during the human experiment. By having that, we showed the reaction time and the action elicited by the squirrel (McCloskey, 1975). By using two sample Z-tests we were able to determine if there is a significant difference between rural and urban squirrel foraging habits. In order to use this specific test you must use proportions, which are percentages, which we had. By using this test we were able to examine the squirrel's response to the stimulus by. In addition, we were be able to create figures to portray our data visually. For example, a graph displayed the amount of time it took for a reaction to occur versus the squirrel's foraging location. With these graphs, statistical significance tests, and observations we were able to show if there is any correlation to a human gene, using epigenetics.

Results

Eastern Fox Squirrel Playback

Squirrel experiment

Written by: Nicole Stewart

Finalized by: Kaitlyn Thrush

           While doing our playback study on Eastern Fox squirrels we found that squirrels in urban environments behave differently than those found in rural, forested environments as shown in a previous study where a correlation was found between density and wariness among squirrels (Parker, T.S. & Nilon 2008). As shown in Figure 1, squirrels that live in urban environments, show less antipredator behaviors than squirrels living in rural environments (Blumstein and Daniel 2005). The differences in behavior observed in the squirrels occurred while they were foraging for food. We believed that because squirrels residing in an urban environment had much more exposure to predatory stimuli, they began to display less of a reaction to stressful stimuli over time. As shown in Figure 2, our hypothesis was correct in the fact that there was a significant difference between urban and rural Fox squirrel reactions toward the playback of the red-tailed hawk (Blumstein and Daniel 2005). In addition we found that Eastern Fox squirrels residing in a rural environment, such as Sanford Natural Area and Baker Woodlot on Michigan State's campus, exhibited greater anti predator behaviors while foraging than squirrels living in an urbanized location because of natural selection, which reduced vigilance activity in squirrels in order to save energy (Blumstein and Daniel, 2005. Mccleery 2009). As shown in Figure 3, 39% of observed squirrels exhibited no alert response to the predatory playback calls. This no response reaction allows the squirrels to save energy by not reacting and transferring the energy toward foraging. This conclusion was decided upon do to the previously conducted the research, "The Loss of Antipredator Behavior Following Isolation of Islands" by Blumstein and Daniel, where kangaroos were isolated from predators on an island, there was a reduced time spent being vigilant toward predators and, as a result through natural selection, their anti predator instincts were diminished, allowing more time for foraging (Blumstein and Daniel, 2005). Rural squirrels are not constantly exposed to stressful stimuli so, when presented with threats of danger, rural squirrel's instinctual reactions were heightened (Ward and Low, 1997). As shown in figure 1, these findings were be portrayed through a statistical analysis known as a two proportion z-test, that generates figures such as those shown in similar scholarly journals (Ward and Low, 1997 and Blumstein and Daniel, 2005).

Squirrel results

Written by: Jordan Fournier

Finalized by: Kaitlyn Thrush

           For this experiment we ran a 2 proportion z-test. We used that test because we used percentages, which are proportions and that is the only statistical test that allows proportions to be used. We ran the test on urban vs. rural using the red-tailed hawk. For the experiment the null hypothesis was that there was no difference in the behavior of the squirrels evoked when we played the call in either urban or rural environments. The alternative hypothesis is that the rural squirrels will react to the predatory call and the urban squirrels will not. After running the test, we obtained a P-value. The P-value ended up being .0000017359. If the P-value is smaller than .05 then we must reject the null hypothesis. Since our P-value was smaller than .05 we accepted the fact that we must reject the null hypothesis. As seen in figure 1, in doing that we accepted the alternative where there was a difference is rural and urban squirrels, and it was a very large significant difference.

Human experiment

Written by: Jacob Trowbridge

Finalized by: Kaitlyn Thrush

           When studying humans we used psychological aspects to understand the results. We predicted that people in an urban setting will not be as alert when crossing a crosswalk compared the people crossing a crosswalk in a rural setting because of the theory of sensory overload states that people in urban environments have so much sensory information coming in that they can not handle it all and that requires them to choose what is most important to them to decode and understand (Wohlwill, 1970). We also predicted that humans living in a rural environment will have increased vigilance instincts because their lack of stressful stimuli will increase one's response when a stimulus is present (Jerison,1965). We believed that humans in a rural environment are not depleting their energy from constantly reacting to stressful stimuli that when a stimulus is presented they will react because of the General Adaptation Syndrome, which describes the the short term and long term responses to stress, established by Hans Selye (Evans, 1982). However, as shown in figure 4, we found no significant difference in the responses between urban and rural human observations. In addition we predicted that ACTH will increase in rural humans causing the reaction of General Adaptation Syndrome, alarm, resistance, and exhaustion, because unless human are adapted to the stressful stimuli around them then these responses will continue as an act of defense (Evans, 1982).

Human results

Written by: Kaitlyn Thrush

Finalized by: Kaitlyn Thrush

           For the homologous human behavior experiment we ran a two sample t-test with the data collected. That specific test was chosen because data from two samples of two populations were collected and we wanted to compare the data to see if they were statistically significant. From the two sample t-test a p-value will be obtained. This value will show any significance within the data, a p-value smaller than .05 will be considered significant and will allow us to reject the null hypothesis. The null hypothesis is that humans will look the same amount, statistically, when crossing the road. The alternate hypothesis is that people in rural areas will look more times before crossing the road, as previously discussed. After running the test, we obtained a p-value of .698966. The p-value is much larger than the .05 that is needed to reject the null hypothesis. We fail to reject the null hypothesis, therefore accepting the null hypothesis; there is not any significant difference between how many times a human will look before crossing the road.

References

Finalized by: Nicole Stewart

Boonstra, Rudy., J.M. Baker, J Castillo, and Q.E. Fletcher. 2007. The Role of Stress Axis in Life-History Adaptations. Pp140-141 in Jerry O. Wolff and PaulW.Sherman, eds., Rodent Societies An Ecological & Evolutionary Perspective. The University of Chicago Press Chicago and London.

Daniel T. Blumstein, Jodie G. Ardron, Christopher S. Evans. 2002. Kin discrimination in a macropod marsupial. Ethology. Issue 9. 108: 815-823.

Daniel T Blumstein and Janice C Daniel. 2005. The loss of anti-predator behaviour following isolation on islands.Proc. R. Soc. B. 272: 1663-1668.

Evans, Gary W.1982. General Introduction. Pp. 3-4 in Gary W Evans, eds, Environmental Stress. Cambridge University Press, New York.

Frid, A. and L. M. Dill. 2002. Human-caused disturbance stimuli as a form of predation risk. Conservation Ecology 6(1): 11.

Jerison, Harry J. 1965. Human and Animal Vigilance. Perceptual and Motor Skills. 21: 580-582.

Kimberley Jayne, Stephen E.G. Lea, Lisa A. Leaver. 2015. Behavioural responses of Eastern grey squirrels, Sciurus carolinensis, to cues of risk while foraging. Centre for Research in Animal Behaviour 116: 53-56.

McCloskey R.J. 1975. Description and analysis of the behavior of the fox squirrel in Iowa. thesis, Iowa State University, 33-38.

McDonnell, M.1.,and S. T.A.Pickett.1990.Ecosystem structure and function along urban-rural gradients:an un-exploited opportunity for ecology. Ecology 71: 1232-1237.

Milgram, S. 1970. The experience of living in the cities. Science, 167, 1461-1468.

Parker, T.S. & Nilon, C.H. Urban Ecosystems. 2008. Gray squirrel density, habitat suitability, and behavior in urban parks. 11: 243.

Provencal, Nadine., 2011. Beyond Genes: Epigenetics Can Stress Modify our Genes?. Mammoth Magazine Issue 9 http://www.humanstress.ca/documents/pdf/Mammouth%20Magazine/Mammoth_vol9_EN.pdf

Robert A. Mccleery. 2009. Changes in fox squirrel antipredator behaviors across the urban-rural gradient. Landscape Ecology. 24:483.

Selye, Hans. 1950. Stress And The General Adaptation Syndrome. British Medical Journal.

Smith, Christopher C. and David Follmer. 1972. Food Preferences of Squirrels. Ecology (Durham). Issue 1. 53: 82-91.

Stanyon, Roscoe., Gary Stone., Monserrat Garcia., Lutz Froenicke. 2002. Reciprocal Chromosome Painting Shows That Squirrels, Unlike Murid Rodents, Have A Highly Conserved Genome Organization. Genomics. Issue 2. 82: 245-249.

Walters, C.J., and F. juanes. 1993. Recruitment limitation as a consequence of natural selection for use of restricted feeding habitats and predation risk taking by juvenile fishes. Can. 1. Fish. Aquat. Sci. 58: 2058-2070.

Ward C, Low BS (1997) Predictors of vigilance for American crows foraging in the urban environment. WilsonBull 109:481,489.

Wohlwill, J.F. 1970. The concept of sensory overload. Division of Man-Environment relations, The Pennsylvania State University. 340-345

Figures
Finalized by: Jordan Fournier

Figure 1. Percent of Time Eastern Fox Squirrels in Both Rural and Urban Environments Spent Foraging and Exhibiting an Alert Behavior. Time percentages of foraging in the observation radius and exhibiting any of the five observed alert behaviors. The total amount of observed time was split into two categories, time spent foraging in the observation radius and time spent exhibiting an alert behavior. The urban environment comprises both urban observations, North Neighborhood and the courtyard by the Union on Michigan State University's campus in East Lansing, MI. The rural environment is compromised by the Sanford Natural Area and the Baker Woodlot on Michigan State University's campus in East Lansing, MI. An inverse correlation between the percent of time spent foraging and the percent of time spent exhibiting an alert behavior is shown between the two environments. Error bars represent one standard error. Written by: Kaitlyn Thrush Finalized by: Jordan Fournier

Figure 2. Alert Behaviors Exhibited by Eastern Fox Squirrels Based on Calls.Scatter plot for the percent of squirrels in each environment, urban and rural, which exhibited an alert behavior in response to the Red-Tail hawk and car horn predatory calls. The rural squirrels exhibited a greater response than the urban squirrels. In the observed rural squirrels a greater response was shown for their natural predator, the Red-Tail hawk. The observed urban squirrels showed a greater response in their unnatural predator, the car horn. The error bars represent one standard error. The data for people interaction was not collect and therefor not applicable. The squirrels were observed on the campus of Michigan State University in East Lansing, MI. Written by: Jordan Fournier Finalized by: Jordan Fournier

Figure 3. Percentages of the most commonly observed squirrel alert reactions. The postures exhibited by the quantified data of all predatory calls performed, Red-Tail hawk and car horn, in both environments, urban and rural. The results presented five different postures exhibited by the observed squirrels. Posture one is indicated by the squirrel on its hind legs, hunched over. Posture two is presented when the squirrel is straight up on its hind legs. A squirrel demonstrates posture three by placing its feet on the base of the tree with its paws pointed upward on the trunk of the tree, the squirrel is looking up in this posture. Posture four is indicated by the squirrel facing down the trunk of the tree with its paws toward the ground and its stomach on the trunk of the tree. The final posture is posture five which is demonstrated by the squirrel laying on a tree branch, with its paws gripped to the underside of the tree and it’s hind legs at the point where the trunk and branch join together. The response of no alert posture exhibited when a predatory call was played was recorded as well. Written by: Jacob Trowbridge Finalized by: Jordan Fournier

Figure 4. The Average Number of Times a Person Looks at a Crosswalk Before Crossing in Rural and Urban Environments. Data has been represented through averages of each environments, rural and urban. The urban location include North Neighborhood and the courtyard by the Union on Michigan State University's campus in East Lansing, MI. The rural location includes the Sanford Natural Area and Baker Woodlot on Michigan State University's campus in East Lansing, MI. The total number of looks was divided by the total number of individuals observed. Urban individuals looked on average 1.83 times before crossing the road where there was no signal, while rural individuals looked on average 1.67 times before crossing. The error bars indicated on the graph show that the urban and rural areas are not significantly different. The vigilance instinct towards vehicles in humans doesn't differ between the environments. Error bars represent one standard error. Written by: Nicole Stewart Finalized by: Jordan Fournier

Figure 5. Squirrel Habituation Documentary Film.