Rabbid(t) Squirrel's Page

      The auditory experience of speech is one of the most complex inputs that a brain must decipher (Ross et al, 2006). On the population level, the articulation of this speech is vital in the communication of information (Ross et al, 2006). However, research done by McGurk and MacDonald has also found that the processing of visual information has a profound influence on the transfer of information. In some cases, the visual perception of information can even override the information that is being communicated verbally (McGurk and MacDonald, 1976). Because of this, the communication and processing of information on the population level plays an important role in the survival and continuation of life in both humans and animals. Natural selection is often impacted by the ability to effectively communicate vital information across various environments. According to Sih, populations and species often times will exhibit behavioral syndromes, which are forms of correlated behaviors that occur across situations. As situations and environments change, behavioral correlations can be adaptive and reflect alternative strategies for survival (Sih, 2004).
      Animals and humans alike have both been known to adjust their behaviors to suit their environment (Sol et al, 2012). For example, animals in urban areas have been observed to spend less time vocalizing about predator threats in order to have more time for other activities, such as foraging (McCleery, 2009). Changes in the geographics of predators can also cause a multitude of behavioral shifts. Because urban and rural areas have different characteristics specifically in the communities of predators, animals have to be able to effectively alter their modes of communication. For example, when captured by a human, urbanized birds have demonstrated different song frequencies than rural birds (Sol et al, 2012). Noise in urban environments has also been observed to alter favored forms of communication. This is the case in many urban environments as noise from human activity can limit the distance at which individuals may efficiently communicate with each other (Halfwerk et al, 2011). Examples of this include the California ground squirrels, which have the ability to shift from low to high harmonics in order to overcome the noise of traffic (Rabin et al, 2003). In addition to this, multimodal signals, which use of more than one form of communication at once, have also been found to be extremely useful in urbanized environments as dual signals ensure that information transferred is not lost in the noise of densely populated areas (Partan and Marler, 2005). However, in urbanized environments, the multimodal enhancements of the eastern grey squirrel suggest that auditory and visual alarm signals are redundant as they each provoke responses in observing squirrels (Partan et al, 2010). Partan specifically found that the grey squirrel relied more heavily on visual information transmitted than verbal. Specific examples of important information that is often transferred across urban and rural environments is the communication of potential predators. Anti-predatory responses to vocalizations have been specifically observed to vary between rural and urban environments in Sciurus niger (fox squirrel) species. In urban settings, fox squirrels have been observed to demonstrate reductions in anti-predator behaviors to both hawk and coyote vocalizations (McCleery, 2009). In previous publications, two forms of anti-predator behavioral communication has been characterized in squirrels. These include visual and verbal forms of communication. In addition to this, these behaviors have been further categorized as calming, vigilant, and alarming (Partan et al, 2010). In fox squirrels, categorized anti-predator behaviors include various forms of scanning and vigilance, tail flicks, tail flags and vocalizations (Partan et al, 2010).
      Like fox squirrels, Homo sapiens (humans) have also been known to adjust their behavior in response to changes in environment and stress. For humans, survival in stressful situations is dependent on the ability to effectively respond to a threatening situations (Taylor et al, 2000). Depending on circumstances in which "fight-or-flight" responses are induced, various forms of communication have been found to be more effect than other. For humans, the formation of social groups has played a key role in survival (Alexander, 1974). Females have specifically been observed to use "tend-and-befriend" mechanisms as a response to stress in social environments. Taylor found that women's responses to stress were heavily characterized by the formation of social groups in order to reduce vulnerability from attacks. It has been suggested that these patterns evolved from the different parenting investments between men and women (Taylor et al, 2000). In addition, humans have also been observed to use evolved mechanisms of communication to call for help. This has been especially true in the evolution of screams. In humans, screaming is one of the most effective forms of communicating danger. Over the years, screams have specifically evolved to signify danger through the perception of increased roughness in the call (Arnal et al, 2015). In addition to this, screams have also been adapted for responders to be able to more accurately locate the the source of the call (Arnal et al, 2015). In a study done by Arnal, they found that the speed of behavioral responses increased as the perceived fear of the scream increased. The use of screams have been significant for the calling of help in solitary locations (Arnal et al, 2015).
      All behaviors displayed by an organism, including that of communication, have an origin in respective genomes. The gene, foxP2 (Foxhead box P2), plays an important role in brain development, through the coding of an important transcription factor (Robinson et al, 2008). This transcription factor plays a key role in the regulation of a variety of genes. Specifically, the foxP2 gene plays an important role in formation of the regions of the brain that are responsible for speech and language. Deficiencies or mutations in the foxP2 gene have been observed to result in stunted articulate communication growth (Enard et al, 2002). The foxP2 gene is predicted present in all mammals, but its location differs greatly. The position of the gene in humans lies on the 7th chromosome (114,086,310-114,693,772) between positions q31.1 and q31.2 (O'Leary et al, 2016). The gene has not been successfully identified in fox squirrels. However, it has been found in other rodents such as mice (Enard et al, 2002).
      The aim of our project is to investigate how social environment can alter the modes of communication used by both Sciurus niger (fox squirrels) and homo sapiens (humans). Based off recent studies of fox squirrel desensitization in urban settings (McCleery, 2009) and the evolution of human screams (Arnal et al, 2015), we hypothesize that social environments will alter the modes of communication used in both squirrels and humans. In order to investigate how communication can be altered, a playback recording that initiates "fight-or-flight" responses in squirrels and humans will be introduced to two different social environments; one solitary and one social environment. For the fox squirrel trials, predation sounds that induced "fight-or-flight" such a red tail hawk and motor vehicle sound will be used along with a white noise sound to act as a negative control. The red tail hawk sound was chosen because it is a common predator of fox squirrels and present in the area of study (Fitch et al, 1946). The motor vehicle sound will be used to elaborate on the previous studies that show squirrel desensitization in busy urban settings (Sol, 2003). We predict that if squirrels are introduced to urban environments, then visual forms of communication will dominate over auditory because in a study done by Partan, they found that in urban environment squirrels more heavily relied on visual forms of communication over that of auditory (Partan et al, 2010). In our study, squirrel responses to predatory sounds will be categorized into several categories including but not limited to: grooming, foraging, quadrupedal vigilance, bipedal vigilance, tail flick, tail flag, general vocalization, tree mounting, and tree ascending. The purpose of response categorization is to determine if fox squirrels communicate more through visual or auditory communication when a threat is perceived. A growing number of studies show behavioral adjustments in urban animals which assist in allowing them to enhance communication within an area of so many disturbances (Sol, 2003).
      In addition to this, we will also be stimulating "fight-or-flight" responses in human through the dropping of a large textbook. Because of the loud and unexpected nature of a book dropping, startled responses in humans will be triggered. We predict that if a human is introduced to a startling stimuli in a solitary environment then they will more heavily rely on verbal forms of communication for help because in a study done by Taylor, they found that screams have evolved to help responders more accurately locate the source of the scream (Arnal et al, 2015). This is extremely beneficial in solitary environments, where help may not be within a visible distance. Because of this, we predict that verbal forms of communication will be more predominate in solitary environment. In order to determine if one mode of communication is dominate over another, we will be organizing the observed responses into categories to determine if visual or verbal forms are more predominate. We will be categorizing human behaviors as visual aggressive, visual defensive, flinching, gasping, and any other vocalizations that hadn't been previously emitted prior to the introduction of startling stimuli.
      Furthermore, future studies will be done to determine if the foxP2 gene is present in both humans and fox squirrels. Because the gene has already been located in humans, future investigations into the gene will primarily be focused on the fox squirrel genome. In the future we would like to use polymerase chain reaction (PCR) to determine if the foxP2 gene can be amplified from the fox squirrel genome. Because the gene has not been successfully identified in fox squirrels yet, primers for PCR will be created off of the gene that has been amplified in mice (Enard et al, 2002). Primers will be based off of mice because of the similarities in genomes since both mice and squirrels are rodents. The purpose of this future study is to determine if the discovered homologous behaviors observed in fox squirrels and humans is connected through genetics, specifically the foxP2 gene.

Methods Written by: Lauren Mileto, Arielle Davidson, Riley Moore, Ben Brown
Revised by: Lauren Mileto, Riley Moore
Finalized by: Ben Brown
      Twenty-five fox squirrels of various sex were tested to see how different predatory vocalizations affect their communicative behavior in urban vs. rural areas. On Michigan State University's campus, the urbanized locations that were tested were outside Wells Hall and in the large field of West Circle near Olds Hall due to the heavy foot traffic present at both locations. We defined heavy foot traffic as ten or more individuals within a 10 foot radius, Monday through Friday. The rural area that was tested for squirrel behavior was the MSU farms of East Lansing. This location consists of vast open space and animals that could contribute to the environmentally adaptive squirrel's way of communicating potential danger. These different locations were tested from the times of 8:00 a.m to 11:00 a.m, Saturday and Sunday. Squirrels were studied at these times because according to Young, that is when squirrels are most active (Young, 2013). Since the urban trials were completed on Michigan State University's campus during the fall 2016 semester, trials were only carried out on the weekend when foot traffic is significantly decreased in order to eliminate any other humans from influencing the responses of the squirrels.
      A playback experiment was utilized in order to determine the anti-predator behavior of eastern fox squirrels (Sciurus niger). Simulated predator calls were used as a means of inducing anti-predator behavior. Recordings of the red tail hawk, from Youtube, were played on an iPhone 6s and relayed through a Bose speaker (Crowe, 2007). The exact same call was used for all the trials. A motor vehicle horn recording, also obtained from Youtube, was used to observe habituation differences between urban and rural fox squirrels (Case, 2013). A third, white noise recording was used as a negative control in order to ensure that the Bose speaker is not inducing alarming behavior in the squirrels. Ten trials for each recording were completed in both the rural and urban locations, along with five trials of the control. A trial number was defined as a squirrel hearing the recording and then producing either an alarm (tail flick, tail flag and vocalization), vigilant (quadrupedal and bipedal) or calm (grooming and foraging) behavior as a response.
      At the start of each trial the date, temperature, weather, and number of squirrels in the surrounding area were recorded. Vocalizations of predator calls were only relayed once squirrels were within 20 meters of the speaker in order to ensure that the calls were heard. This distance was pre-defined by markers placed in the ground. Investigators observed the behavior from a safe distance away, behind either a tree or boulder. Limited stimuli ensured that the responses of the squirrels were the result of the playback and not outside stimuli. One investigator observed the squirrels with binoculars while the other wrote down the visual and vocal responses observed. Data was focused on individual squirrels, however observations of squirrels in the area were also recorded if applicable.
      Squirrel observations were recorded after playing three predatory vocalizations from a bose speaker. The information that was obtained from the observation was the response, the type of response (auditory or visual), and the frequency of that response. For each trial, every aspect was kept constant except for the location of squirrels which was the variable being examined. After all data was collected, squirrel's communicative language in rural and urban areas was determined. A chi-squared test was chosen to test the statistical significance of our data. The test was run on various aspects of the results. A p value 0.05 was deemed statistically significant, which means that there is strong evidence that the null hypothesis is not true and the results are actually significant and not due to random chance. We compared the data between the rural and urban responses when a hawk sound was introduced, and the rural and urban responses when a car sound was introduced. Standard error of the data was also found which gives the experiment validity. Error could occur if the results that were obtained were due to chance instead of the treatment, which is the predation playback sound.
      Thirty human test subjects were observed under enhanced stress conditions through the facilitation of acoustic startling stimuli. Startling stimuli was administered through the surprised dropping of a chemistry textbook of over 800 pages. Responses were compared across social and solitary settings in order to better understand how the visual presence of multiple humans impacted the communication of danger. Two separate social situations were designed to carry out the treatment and control experiments. Both the social and solitary human experiments took place in the main hallway located in Holmes hall on Michigan State University's main campus. The main hallway was defined, going from west to east, as the space between classrooms C-106 and C-101. Space inside of the classrooms and that of Sparty's were excluded from the study. Two different common class transition times were used during the day for the social control and treated replicates. Trials were carried out between 10:00-10:20 AM and 12:20-12:40 PM Monday through Friday, only when classes were in session. The social environment was defined as having more than ten people present in the hallway excluding the experimenters. The more solitary environment was carried out during quiet hours after 9:00 PM Monday through Thursday in the same location of Holmes Hall. Fridays through Sundays were excluded due to the variable nature of students being in the hallway during the night hours then. The solitary environment was defined as having fewer than five people in the hallway, including the two experimenters and the test subject.
      In the social setting replicates, a chemistry textbook that was over 800 pages was dropped by one of the experimenters once they were within ten meters of the test subject. Due to high social contact at that given time, only the reaction of the person closest to where the textbook was dropped was recorded. The reaction was recorded by the second experimenter who observed from a seated position on the benches located on the north side of the hallway. The observation of the reactions to startling stimuli in solitary settings was administered the same way as the social setting except for the fact that fewer than five people, including the experimenters, were present. Unlike the squirrel replicates, only one sound was used as a treatment because the primary focus of this aspect of the lab was to observe how danger is communicated across various social settings in comparison to how fox squirrels communicated the presence of predators. Negative control replicates were also designed in order to ensure that the presence of the experimenters prior to dropping the textbook was not influencing the response of the subjects. Normal chatter was carried out around subjects to ensure a response was not influenced in the hall during both social and solitary settings.
      Fifteen replicates for the social and solitary setting were carried out (30 in total) with the addition of 5 negative control replicates for each setting (10 in total). A replicate consisted of one subject's response(s) to the startling stimuli that was facilitated through the dropping of the chemistry textbook described above. The primary responses that were recorded were vocal and visual language. Any kind of vocal language that was said or yelled in response to the treatment was recorded as auditory. Specific visual communication such as defense and aggressive type postures were recorded. Defense types postures included covering the face with hands and making oneself small (such as pulling arms inward or crouching down). Aggressive responses including making oneself larger, such as standing taller, and squaring shoulders was recorded. On top of the visual defensive and aggressive responses, flinching was also recorded. If no outerward auditory or visual responses were observed that was recorded as well. A chi squared test was designed to analyze the frequency of each response in varying social settings. The various reflex responses were categorized as vocal, visual, a combination of vocal and visual, and no response were compared to solitary and social settings. In the chi square, for each setting there were 14 degrees of freedom, points were considered significant if the calculated probability (p-value) was 0.05.
      The foxP2 gene found in the fox squirrel and human genome was amplified via PCR. DNA was obtained from the hair follicles of a fox squirrel. The DNA was extracted from the follicles with the use of enzymatic laundry powder, which consists of protease, amylase, surfactant, propylene glycol, and water (Guan, 2013). Hair samples were cut into 2 mm pieces and digested in 100 microliters (uL) of extraction reagent (pH 10.3) for 90 minutes at 50 degrees Celsius. The extraction reagent contained 3 milligrams (mg) of enzymatic laundry powder and 1x PCR buffer (20 millimolar (mM) Tris-hydrochloric acid, 20 mM Potassium chloride, 10 mM Ammonium Sulfate, and 1.5 mM Magnesium chloride). The sample was than heated to 95 degrees Celsius for 10 minutes for optimal extraction. DNA was stored at -18 degrees Celsius until usage.
      After extraction, PCR reactions were carried out in 20 microliter (uL) increments. Four replicates were created, and a total of 80 uL of PCR reaction were created. Forward and reverse primers were designed using the foxP2 gene sequence in a house mice identified by the National Center for Biotechnology Information database. The primers F (anneliating at 1,088 base pairs to 1,107 base pairs on the mouse foxP2 gene) and R (anneliating at 1,899 base pairs to 1,918 base pairs on the mouse foxP2 gene) will be used to amplify a product that will be approximately 3,000 base pairs. The PCR procedure was based off of an experiment done by Guan (Guan, 2013). Reactions were carried out in 20 uL reaction volumes that each contained 0.5 unit HotStar Taq DNA Polymerase, 200 micromolar (uM) dNTP mixture, 1x PCR buffer, and 200 nanomolar (nM) of each primer. Samples and reagents were kept on ice until usage. The samples were placed in a Labnet thermocycler for amplification. Reactions were denatured for 10 minutes at 95 degrees Celsius and then put through 35 cycles of 95 degrees Celsius of 20 seconds each. Annealing was carried out for 40 seconds at 62 and 57 degrees Celsius. Extension was carried out at 72 degrees Celsius for 1 minute followed by a final extension at 72 degrees Celsius for 10 minutes (Guan, 2013). Amplified DNA was pipetted and confirmed through gel electrophoresis (Guan, 2013). The agarose gel was made with 0.35 g of agarose powder and 35 mL of SB buffer. The agarose gel was poured into a gel tray and one comb that produced 12 wells was inserted. The gel was allowed to sit for about 20 minutes in order to solidify. Once the gel had completely solidified, the comb was pulled out and sodium borate (SB) buffer was pour over the gel until it reached the fill line on the apparatus. Three uL of DNA ladder was added to the first well. Three uL of the PCR reaction was mixed with one uL of loading dye and then added into the following wells. One sample from each PCR reaction was run. A total of four samples were checked in the gel. An electric current was run through the gel for approximately 14 minutes. Following, the gel was checked for DNA bands under UV light. The bands shown in the non-control wells were examined and identified to confirm that they do have the correct base pair lengths that correspond with foxP2. A band should be present around 3000 base pairs if the foxP2 gene is successfully amplified.

Predicted and Experimental Results

Written by: Lauren Mileto, Arielle Davidson, Riley Moore, Ben Brown
Finalized by: Lauren Mileto
      We predict that urban squirrels will use less auditory and more visual signals in response to a predatory stimuli compared to rural squirrels. This will occur because the heavily populated areas of an urban setting are very noisy and make it difficult for squirrels to effectively communicate through vocal cues (Partan and Marler, 2005). Due to this difference in environments, we predict to observe rural squirrels using more vocal communication than urban fox squirrels (Partan and Marler, 2005). The data collected thus far in our study follows along with our predicted results. Fifteen trials for both urban and rural fox squirrels have been completed (Figure 1). Based off the currently obtained data, we found that six of the fifteen rural squirrels responded to the predatory stimuli with vocalizations while only two of the fifteen urban squirrels used vocalizations (Figure 1). We predict that the urban squirrels will use various visual methods of communication such as tail flags, tail flicks, bipedal vigilance, or quadrupedal vigilance which were observed and categorized by Partan (Partan et al, 2010). We predict that the urban setting squirrels will spend more time foraging because McCleery's study showed that squirrels are less responsive to predator vocalizations and spend less time participating in vigilant behavior due to the redundancy of predator stimuli they are presented with in the urban setting (McCleery, 2009). The trials thus far support our predictions by showing that two trials of urban squirrels responded with a foraging behavior while zero of the rural squirrels responded in this way (Figure 1). Overall, we predict that our current trials will follow the same trend based off the previous pieces of literature contributing to the study of fox squirrel communication (McCleery, 2009).
      We predict that humans will respond with more acoustic signaling, such as calling out for help, when alone rather than in social groups because of the need to communicate to others, who may not be in visible distance, for help (Arnal et al, 2015). A study done by Taylor et al (2000), found that women would specifically surround themselves in social groups as a way to respond to stress. However, when the protection of a group was not available, the use of acoustic signaling became more imperative. The evolution and use of screaming has played a key role in the signaling of danger, especially when fight or flight instincts are not an option (Arnal et al, 2015). Over time, the specific roughness of screams has been selectively adapted to signify danger across varying communication types (Arnal et al, 2015). Other selective advantages that have evolved included screams to be perceived more as fear-inducing and the ability of calls to increase the speed and accuracy for locating the source of the cry (Arnal et al, 2015). This evidence strongly supports our prediction that humans in solitary environments will more heavily rely on screams due to the seclusion of the environment and the need to broadcast signals over longer distances. The data collected thus far from our human experiment further supports our prediction that in solitary environments people are more likely to respond with vocal communication. Out of eight replicates, in the social setting, four responded with visual communication, two vocal, one a combination of both, and one had no response (Figure 2). In the solitary setting, five responded with auditory while only two were visual, making the auditory responses more common compared to the visual ones (Figure 2).
      We predict that the results of our DNA extraction and PCR experiment will show that the foxP2 gene is present in the fox squirrel genome because of the research done by Wolfgang Enard, which found that most mammals including chimpanzees, apes, and mice have the gene present in their genome (Enard, 2002). We predict that if we are able to successfully amplify the foxP2 gene, then we will see a band that is around 3,000 base pairs long on a gel because the section of the gene that we are trying to amply based off of foxP2 gene sequence amplified in mice is approximately 3,000 base pairs long (Enard, 2002). The predicted results of our gel electrophoresis would be similar the results obtained by Yates and his colleges (Figure 3). The first well will be a ladder marker, to act as a positive control, while the next four will be filled with purified DNA samples from the fox squirrel hair follicles combined with a dye that would react with UV light and present a glow around 3,000 base pairs (Figure 3).
      We predict that urban squirrels and social humans will use more visual cues when communicating danger to others because they communicate more efficiently with visual signals in noisy environments (Partan et al, 2010). We predict that humans communicate similarly to squirrels when noise is present because when a human uses visual movements, the listener's ability to understand what they are trying to communicate is improved, especially under noisy environmental conditions (Ross et al, 2006). Humans use visuals to warn others of potential danger in busy environments as well. Real data was collected on responses of urban squirrels, rural squirrels, social human, and solitary human. Out of the fifteen trials done for urban squirrels, eleven responded with a visual response, two responded with an auditory response, and two continued to forage, which was categorized as a no response (Figure 4). Out of fifteen trials for rural squirrels, eight responded with visual signals, six responded with auditory, and one responded with both (Figure 4). For eight trials of social humans, four responded with visual signals, two responded with auditory signals, one responded with visual and auditory signals, and one had no response (Figure 4). For eight trials of solitary human, two had a visual response, five had an auditory response, and one had a visual and vocal response (Figure 4). This data supports our predictions showing that urban squirrels and social humans use more visual signals while rural squirrels and solitary humans use more auditory signals to communicate danger. We predict that very few squirrels and humans will respond with both visual and auditory behaviors because using both signals together has proved to be redundant (Partan et al 2006). This is similar to the data that has been collected, since very few responses of auditory and visual responses have been recorded.