Squirrels are a unique species that can live in urban and rural environments, adapting their lifestyle to locate food and shelter (McCleery et al, 2008). A species common to Michigan State University's campus, MSU, is the eastern fox squirrel with the scientific name Sciurus niger (Salsbury, 2008). They tend to locate themselves in leaf nests high up in deciduous trees, also known as drays (McCleery et al, 2007). Drays offer protection from aerial and terrestrial predators as well as provide warmth throughout the winter (McCleery et al, 2007). Squirrels often become prey to many predatory species such as the hawk, raccoon, great horned owl, and snakes (Thorson et al, 1998). To avoid these different predators, they often exhibit flight behaviors, like running to the nearest tree, running away, or taking cover under brush in times of danger (Thorson et al, 1998). When threats are not present squirrels are often found foraging (looking for food) or eating. Squirrels reacting aggressively would charge the predator or hold ground (Nunes, 2014).

Behaviors can be described as being instinctual or learned. An instinctual behavior is an action performed when a stimulus creates reactions in the nervous system to form spinal reflexes (Lashley, 1938). These reactions are genetically coded and inherited from generation to generation; meaning individuals are born with this reaction (Panksepp, 2000). Eastern fox squirrels, in a wide variety of habitats, all exhibit behaviors of food hoarding, this would be an example of instinctual behaviors (Steele et al, 2006). Behaviors which develop over time, through observation, teaching, or experiences are learned behaviors (Lang et al, 2000). Learned behaviors can result from acquired fears or desensitization of past fears (Lang et al, 2000). An example of a learned behavior is how eastern fox squirrels, in urban areas, no longer view humans as a threat. They have adjusted to living in highly human populated areas through an evolutionary process, referred to as suburbanization (McCleery et al, 2007). All reactions, both learned and behavioral, are induced by an organism experiencing fear or arousal (Panksepp, 2000). These emotions are internalized through a neural network and activates the defense system, which determines the mobilization of an individual (Lang et al, 2000). The defense system has automatic motor outputs called defense action, also known as fight or flight (Lang et al, 2000).

A common term in biology and psychology, known as fight or flight, refers to the options that an organism has when presented with a potential threat (Lang et al, 2000). The organism will instantaneously choose to attempt to fight for survival or try to escape the perceived threat (Lang et al, 2000). Cannon's Theory, which states the Thalamus of the brain perceived emotional signals which triggers a signal cascade, helped to develop modern research on instantaneous reaction in organisms to environmental stimuli (Cannon, 1929). A cascade begins when part of the nervous system (sight, smell, sound, etc.) detects danger. This trigger will send a message to the organism's adrenal gland (Cannon, 1929). The adrenal gland will produce epinephrine, more commonly referred to as adrenaline, to provide the organism a boost of energy to respond to the threat (Cannon, 1929). The "startle response" is the term for this emotional-chemical reaction to potential threats (Lang et al, 2000). Squirrels and many other species have an adrenal gland which is regulated by the phenylethanolamine N-methyltransferase gene, or PNMT (Hoehe et al, 1992). This gene is located on chromosome 17q21-22, with the base pairs AATAAA (Hoehe et al, 1992) (Batter et al, 1988). The PNMT gene that squirrels carry is an ortholog of the PNMT gene which 150 species of mammals share, including humans (Hoehe et al, 1992). The gene codes for the terminal enzyme responsible for the catalyzation of epinephrine in both humans and squirrels (Hoehe et al, 1992). The linked gene between humans, squirrels, and 148 other species means the signal cascade between the organisms with the gene will be similar when a threat is identified (Hoehe et al, 1992).

Since squirrels are common all over the country, they encounter a variety of different predators. We are investigating whether or not squirrels will react to predators that are not present in their habitat. Our experiment specifically addresses the question: are the reactions of eastern fox squirrels to predators a learned or instinctual behavior? To test this, observations of on-campus squirrels and their reactions to both off-campus and on-campus predator playback will be recorded, then the PNMT gene in squirrels and humans will be investigated to find similarities and differences. This similarity will be observed further by an observational study on how humans react to a potential predator, spiders. We hypothesize that the fox squirrels will exhibit similar reactions in an off-campus predator playback, the unconditioned threatening stimuli, to the on-campus predator playback, conditioned stimuli. We predict the squirrels will react to predatory noises instinctively because the unconditioned stimuli, the off-campus predator, should induce stress proteins to trigger neural activity which results in defensive behaviors (Slos and Stoks, 2008). We hypothesize our three experimental test sites on campus will have the same reactions from the observed fox squirrels. We predict the different test sites will be statistically similar in squirrel reactions because the locations on campus are analogous in habitats (Cavallini and Nel, 1990). We hypothesize the human reaction of fear to spiders is a learned behavior, and will be reflected during our trials with less fight or flight reactions. We predict human fear towards spiders will be a learned response because not everyone will react identically (Panksepp, 2000). We hypothesize the controls in either of our experiments, human and squirrel, will not elicit any response in our subjects. We predict the controls in our experiment will result in no response because there is no potential threat (Ulagaraj and Walker, 1973).

Methods

Finalized by: Nicholas Yeager

We observed eastern fox squirrels, at three locations on campus: between Jenison Field House and McLane Baseball Stadium (L1), center of Brody complex (L2), and behind the Main Library on the north river bank of the Red Cedar River (L3). Each of these locations are similar in human traffic, squirrel populations, and tree density. Our playback experiment included a Polaroid PTB3005 Bluetooth speaker and an Apple iPhone 6plus to provide the predator calls at 6000Hz. We played a red-tailed hawk cry (on-campus predator) and a bobcat call (off-campus predator), recorded by Robert C. Stein (Stein, 1961). The speaker will be placed on the ground at each location: L1, L2, and L3, in a squirrel populated area. We recorded each trial on an Iphone 5s and reviewed it later to record data.

Squirrel reactions were recorded using a number key: 1-acting aggressively, 2- eating, 3-Foraging, 4-alert, 5- runaway, 6-tree themselves, 7-taking cover, 8-doing nothing, and 9-other (Keller, 2014). We grouped each of these responses into three categories: ignore, flight, or fight. Ignore included the behaviors of ignoring, eating, foraging, and not responding. Flight included behaviors of alert, runaway, treeing themselves, and taking cover. Fight consisted of aggressive behavior towards the sound. We waited 15 minutes before starting trials so squirrels do not associate us with predatory calls (Schwagmeyer and Brow, 1981). We observed squirrels from 11 A.M. until 2 P.M. on Mondays and Tuesdays during the months of October and November in 2016. With our squirrel experiment, we began each trial by placing the speaker at each test site (L1, L2, L3). For our control, our group recorded observation of the squirrels when the speaker played no sound. The squirrel experiment control was negative, as we didn't expect them to react to the speaker at each test site.

We allowed 15 minutes following each trial for the squirrels to relax (Schwagmeyer and Brow, 1981). After our reset period, we played a predatory sound for a period of thirty seconds (Schwagmeyer and Brow, 1981). Following the reset period, we repeated our trial with the other predatory sound. At each location, the order of the predatory calls were chosen at random. We recorded data at 10 second intervals, beginning 10 seconds prior to the predatory playback call and through the 30 seconds of predatory call (Schwagmeyer and Brow, 1981). We performed five trials of each predator and control at all three locations and 30 human trials. Two group members filmed the squirrel's, one played the predator playback sounds, and the fourth cued everyone when to start and stop cameras and playback sounds.

Our human experiment was performed at the main Library at MSU to determine if fears are instinctual or learned (See L4). A fake spider was placed on the sidewalk leading into the library. Again, group members recorded observations from a distance. We observed three different human reactions to our experiment: fight, flight, or ignore. A fighting reaction was categorized as stomping, kicking, and anger (Baron and Richardson, 2004). Flight was categorized as running, screaming, or backing away from the fake spider (Taylor et al, 2000). An ignoring reaction was no visible signs of distress or concern after seeing the spider (Taylor et al, 2000). The negative control was observing people walking in and out of the library with no spider present. Following the observational period for both experiments, we analyzed our data using the chi-square test to check the frequency and validity of our data. The chi-square test fits our data because it is mutually exclusive, random, and has a large sample size (Roscoe and Byars, 1971). We used the chi-square test of independence to ensure our three test sites and the reactions to on-campus and off-campus predators were statistically the same. For our human experiment we used chi-square test of independence to test if the observations of fight or flight response are statistically different from the observed no response category.

Results

Finalized by: Ashley Kimmel

The three test sites used for the eastern fox squirrel experiment were at Brody Complex, the field between Jenison Field House and the Drayton McLane Baseball Stadium, and the spillway behind MSU's main library (figure 1 A). The bobcat playback (off-campus predator) had 100% reaction in 10 trials, five at each location, Brody and Jenison. At the library, four of the five trials had reactions to the bobcat (figure 1 B). The chi-square test proved these locations were statistically the same for the bobcat playback with a X^2 value of 2.143 and a p-value of 0.3425 (p>0.05). The red-tailed hawk playback (on-campus predator) had 100% reaction in 10 trials, five at each location, Brody and behind the library. Jenison Field House had 4 of the 5 squirrels react to the playback (Figure 1 C). With a X^2 of 2.143 and a p-value of 0.3425 (p>0.05) these locations were statistically the same. The negative control was the speaker with no sound and at every location. 100% of the squirrels did not respond to the control (Figure 1 D). The chi-square test proves all locations are statistically the same with a X^2 of 0 and a p-value of 1 (p>0.05). Since every location was statistically the same, we were able to combine all locations into one.

With all locations cumulated, the playback of the two predators were compared for off-campus and on-campus responses for eastern fox squirrels. The bobcat playback had 14 out of 15 squirrels respond. The red-tailed hawk was the exact same with 14 out of 15 trials reacting. In both cases, only one out of the 15 trials did a squirrel not respond to the playback (Figure 2). The X^2 value for these two was 0 and had a p-value of 1 (p>0.05). This means the squirrels' reaction to the two predators, off-campus and on-campus, were statistically the same.

Since both the predators and locations were statistically the same, we grouped together the results for the bobcat and red-tailed hawk trials at all the locations to compare playback to the control (no sound) trials. When predator playback was used 28 out of 30 squirrels reacted. This was divided by two to keep the ratio 1:1 between the playback and control trials. By doing so, 14 out of 15 squirrels responded and only one didn't for the predator trials. In the control at all locations, none of the 15 squirrels responded to the speaker with no sound, 100% of the squirrels ignored (Figure 3). When comparing the predator playback to the control, the X^2 value was 26.25 and the p-value was 3E-7 (p <.05). This means the results from squirrels reacting to the predators and the control were statistically different.

For the human experiment, 30 subjects were used for both the fake spider trials and the control trials (no spider). When the fake spider was present, 10 out of 30 people reacted with either fight or flight, while 20 ignored the spider's presence. Without the fake spider, every individual remained calm when entering or exiting the library; 100% of the trials had no response (Figure 4). The X^2 value is 10.588 and a p-value of 0.0011 (p <.05) meaning the potential threat and control were statistically different.

References

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Figures

Finalized by: Lindsie Egedy

Figure 1- (A) Test sites for squirrel and human trials. These locations were selected because of similar human traffic flow and tree density in all areas. Sites L1, L2, and L3 were used for fox squirrel trials. L1 was the Brody complex, L2 was the field between Jenison Field House and the Drayton McLane Baseball Stadium, and L3 is the spillway behind the library. L4 was used for the human experiment and it was the back entrance to MSU's main library. (B) Comparison of fox squirrel reactions to bobcat playback at the three test sites. A stereo played a bobcat growl to simulate an off-campus predator. This was repeated at three locations with 5 trials at each site. Observations were taken of individual squirrel's behavior when the bobcat sound was played. Observations were categorized as response and no response. The test sites proved to be statistically the same with a X^2 value of 2.143 and a p-value of 0.3425 (p>0.05). (C) Comparison of fox squirrel reactions to red-tailed hawk playback at the three test sites. A stereo played a red-tailed hawk cry to simulate an on-campus predator. This was repeated at three locations with 5 trials at each site. Observations were taken of individual squirrel's behavior when the red-tailed hawk sound was played. Observations were categorized as response and no response. The test sites proved to be statistically the same with a X^2 value of 2.143 and a p-value of 0.3425 (p>0.05). (D) Comparison of fox squirrel reactions to the control. A stereo was placed in the site locations with no sound. This was repeated at three locations with 5 trials at each site. Observations were noted for individual squirrel's behavior in the presence of the stereo. Observations were categorized as response and no response. The test sites proved to be statistically the same with a X^2 value of 0 and a p-value of 1 (p>0.05).

Figure 2- Comparison of fox squirrel reactions to hawk and bobcat playback calls. This is a cumulation of the three test sites (L1, L2, and L3) because they are statistically the same. We used a stereo to play an off-campus bobcat growl and on-campus red-tailed hawk call. We did 5 trials at each location, which adds up to 15 trials for each predator call. Observations of individual squirrel behaviors were taken when a call was played for 30 seconds. Observations were categorized as response and no response. The response to both the off-campus and on-campus predators were statistically the same with a X^2 value of 0 and a p-value of 1 (p>0.05).

Figure 3- Percent of fox squirrel reactions to predator playback compared to the control. This is a cumulation of the predator playback at three test sites because they are statistically the same. The stereo was used to play the bobcat growl and red-tailed hawk cry. In the control, the speaker with no sound was used to ensure the squirrels weren't reacting to the speaker. Observations of response or no response were taken of the squirrel's behavior during playback or no sound. Using a chi-square test, we prove the predator call was statistically different from our control with no sound. The X^2 value was 26.25 and the p-value was 3E-7 (p<0.05). Predator playback and our control were statistically different.

Figure 4- Comparisons of human reactions to a potential threat of a fake spider to no spider. A fake spider was placed on the sidewalk leading into the back entrance of MSU's main library (L4). Observations of response and no response were taken of people who noticed the spider. In the control, with no spider present, observations of people walking in and out of the library were taken. The X^2 value was 10.588 and a p-value of 0.0011 (p<0.05) meaning the potential threat and control were statistically different.

Figure 5- A documentary of learned vs. instinctual anti-predator behaviors for squirrels and humans.

Appendix