Final Author: [B-123], [B-070], [B-207], [B-680]

     The purpose of this observational study was to explore and compare satiation-response behaviors between humans and fox squirrels and investigate a possible genetic link. Over the course of two months, our team observed squirrels and humans around the campus of Michigan State University and analyzed the satiated behaviors they exhibited. We hypothesize that squirrels and humans exhibit satiation after foraging or smoking because these behaviors stimulate the release of dopamine which is received by dopamine receptors, for which the DRD1 gene is vital to the formation of, and translated into observable feelings of satiation (Misener, 2004). We predict that more satiated behaviors will be observed from squirrels when they are actively foraging based on Trevor Poole's 1997 discussion of animal wellbeing. Studies investigating the release of dopamine while smoking lead us to predict that more satiated behaviors will be observed while humans are actively smoking (Patel, 2011). PCR was used to amplify a target sequence of DRD1 from human saliva samples. We predict to find evidence of DRD1 in humans because we chose previously successful primers and used multiple purified samples (Ostadali, 2004). The amplified DNA was analyzed using a gel electrophoresis test against the GoldBio 100bp ladder. While previous publications exemplify cause-effect relationships between the release of dopamine and satiation responses in humans and squirrels, our studies attempt to contribute a comparison of the responses and develop a more advanced genetic connection through analysis and investigation of the DRD1 gene.

     Final Author: [B-123]

     The first fox squirrels (sciurus niger) in Michigan were sighted by Norman A. Wood in 1875, outside his home in Lodi Township, Washtenaw County (Wood, 1922). In the late 1800s-early 1900s, sighting a fox squirrel in Michigan was rare as they were often overshadowed by the abundance of northern gray squirrels in the area, since gray squirrels preferred heavily wooded habitats. However, as deforestation became increasingly common, the population of gray squirrels died down (Wood, 1922). In a study conducted on quadrupedal animals, Robert Kennicott (1857) discovered that the preferred habitat of the fox squirrel is known as oak openings (Kennicott, 1857). These oak openings formed when the glacial lakes; Lake Wayne, Lake Warren, and Lake Lundy drained away as a result of the watershed created during the late Cenozoic era (Forsyth, 1960; Larson and Schaetzl, 2001) and produced sandy soils on which trees would grow (Comer et al, 1995). Comer's (1995) study was focused on constructing digital maps which illustrate pre-settlement vegetation in Michigan with the goal of raising awareness for biodiversity in Michigan and influencing views on land management. Comer's (1995) maps show that a large section of Michigan land are oak openings which correlates with the large population of fox squirrels. Since fox squirrels have become more common, it is important that we understand their behaviors in pursuit of a healthier and safer coexistence between humans and the fox squirrel.

     A 'happy animal' described by Trevor Poole (1997) in his experiment on Eastern grey squirrels found that an animal who is alert and busy and is able to rest in a relaxed manner (Poole, 1997). The cognitive state of fox squirrels is greatly dependent on the social factors and the physical environment around them (Poole, 1997). For example, among the physical environment, loud and sudden noises can cause disruption in their mood (Poole, 1997). A study done by La Morgia (2017) found that squirrels that live around more people were more approachable than the ones who lived in the woods with no human contact. This can be observed through the fox squirrel's alarm system which involves different degrees of specificity in tail signals. Twitches are general; they have no significant stimulus-signal association with predator appearance, manner of approach, or their combinations (Poole, 1996). A moderate stimulus-signal association (such as tail movement) with predator type occured when both appearance and manner of approach differ (McRae and Green, 2014). Compared to squirrels, humans have a more advanced language of expression. In an effort to conduct an evolutionary approach to Charles Darwin's findings on human emotion, a study was conducted by Matsumoto (2008) and colleagues to draw together recent studies on humans facial expressions with Darwin's theories. This study was founded on the notion that facial anatomy expresses a lot of the emotions that a human feels, like when humans feel a sense of joy or happiness their eyes sparkle, wrinkles around their eyes are formed and the corners of the mouth get drawn backward (Matsumoto, 2008).

     Researchers Felce and Perry conducted studies on humans in which they defined satiation as the degree of an individual's perception of their fit between their needs or aspirations and their objective situation (Felce & Perry, 1995). Trevor Poole studied the importance of animal wellbeing and happiness in small mammals, i.e. squirrels (Poole, 1996). Poole concluded that small mammals exhibit their state of satisfaction and satiation as being alert, busy, relaxed, confident, and does not show otherwise abnormal behavior (Poole, 1996). In contrast, squirrels that are agitated or in distress will dart back and forth to warn off potential predators (Gibbens, 2017). Behaviors linked to the release of dopamine in squirrels include most notably collecting and storing nuts, two actions that help ensure this species survival (Moller 1983). Our team predicts that these behaviors are linked to satiated behaviors.Utilizing some of the observational methods and techniques from Sarah Partan and her colleague's in their study of gray squirrels in urban and rural areas, our team investigated eastern fox squirrels on Michigan State University's campus in both 'urban' and 'rural' areas. Our study defined these areas as 'busy' (Beaumont Tower, Michigan State University, East Lansing, MI) and 'secluded' (Beal Garden, Michigan State University, East Lansing, MI) to examine any potential differences in satiation behavior across differing environments (Partan et al., 2010). In order to test for a homologous behavior in humans, our team focused specifically on the satiation responses of humans during and after the use of electronic cigarettes. As defined by Matsumoto (2008) who analyzed the findings of Charles Darwin (1872) and found that human satiation can often be observed as lip protrusions, partial closure of the eyelids, raising of the upper lip if contempt, eyes sparkling and the skin under the eyes being wrinkled if happy, and the zygomatic and orbicularis muscles contracted when joyful (Matsumoto, 2008). We used these noted human expressions to attempt to quantify human satiation responses during and after the use of electronic cigarettes (E-cigarettes). Our team hypothesizes that more satiated behaviors will be observed after the squirrels and humans perform a known dopamine releasing behavior (foraging and smoking respectively). From our hypothesis we predict that more satiated behaviors will be observed from squirrels in rural areas versus urban areas.

     Because we studied satiation based upon known dopamine releasing behaviors, we decided to investigate the similarities in the reward and pleasure systems in our two test species. We discovered that humans and rats (close relative of squirrels) share a family of genes, the Dopamine Receptor (DR) family, that code for dopamine receptors in the brain. Dopamine receptors are abundant in and vital to the function of reward system structures such as the Nucleus Accumbens (NAcc) and Ventral Tegmental Area (VTA) in humans (Lee et al, 2006)(Malenka et al, 2009). We investigated the Dopamine Receptor D1 (DRD1) gene specifically because it is the most abundant of the family in these parts of the brain (Weiner et al, 1991). The known dopamine releasing behaviors that we studied were the collection and processing of food by squirrels and nicotine intake by humans. Both actions stimulate the release of dopamine and therefore the stimulation and use of dopamine receptors in the respective species (Moller, 1983) (Comings et al, 1996). Given that we were not able to find concrete and explicit evidence of DRD1 presence in squirrels as we could in humans, we attempted to bolster our assumption of DRD1 presence in squirrels by conducting DNA purification, PCR amplification, and gel electrophoresis testing for DRD1 gene on both human and fox squirrel tissue samples. Our team hypothesizes that both humans and squirrels would carry the DRD1 gene based on previous PCR diagnostic testing (Ostadali et al., 2004;Lee et al., 2006).

     Final Author: [B-207]

     Fox Squirrel Observation

     Materials:

• Canon EasyShot
• Canon 60D DSLR
• GoPro Hero 5 Black
• iPhone 8+
• iPhone 7

     Fox squirrels were observed performing their defined satiation behaviors at two locations on Michigan State University's campus that varied in their business and foot traffic (Partan et al., 2010). These were Beal Garden south of the Main Library and the area surrounding Beaumont Tower. No observations were made during poor weather conditions such as heavy rain or snow. Observations were focused to a single squirrel at a time by a videographer using one of the cameras listed above. The video was later analyzed for potential dopamine releasing behaviors and satiation related behaviors. Observations were made as frequently as possible though totaling at least ten minutes per week from late September to early November 2019. Higher resolution cameras were used when possible though most videos were taken by team members with their personal cell phones. Most observations were made in the evening when squirrels were more active (Partan et al., 2010).

     Human Observation

     Human observation methods mirrored a previous study on smoking performed by Vimal Patel and his team in 2011 that observed humans in a satiated state (Patel, 2011). Two observers positioned a GoPro Hero 5 and a Canon 60D near the intersection of M.A.C. Avenue and East Grand River Avenue in East Lansing, Michigan on either side of the street to record every person that walked through the sampling frame (Patel et al, 2011). This study area was chosen due to its close proximity to multiple smoke shops and high pedestrian traffic flow. Observers positioned themselves in a way that would not alert subjects in the frame. Times that smoking behavior was observed were logged in the field and analyzed later (Patel et al, 2011). The sampling frame was altered to not include people in vehicles and children. Children were defined as anyone who appeared to be twelve years old or younger. Smoking was defined as anyone observed holding an e-cigarette in their hand or mouth.

     DNA Purification

     Materials:

• NanoDrop Spectrophotometer
• Microcentrifuge
• Hot Water Bath
• Bio-Rad Chelex-100 Molecular Biology Grade Resin
• Deionized (DI) Water
• 15 mL Centrifuge Tubes
• 2 mL Microcentrifuge Tubes

     Purification methods were copied directly from IDT's Cheek Cell mtDNA Isolation and Purification though similar methods were found in David Sweet's work on DNA extraction (Sweet et. al, 1998). Human saliva samples were collected from multiple participants before being poured into a 15 mL sterile tube to settle for 15-20 minutes. Samples were aliquoted into 2mL microcentrifuge tubes and spun at full speed for about 5 minutes to allow the cells to cluster at the bottom of the tube. Excess liquid was drained off and 100uL Chelex resin was added to each tube. The tubes were then placed in a hot water bath for 10 minutes then placed on ice for about 3 minutes before being centrifuged again to pull out the chelated contaminates. DNA containing liquid was then transferred to clean tubes and placed on ice. DNA concentration in the purified sample was measured using a NanoDrop spectrophotometer. Samples were frozen between uses.

     PCR

     Ingredients and respective concentrations were derived from PCR Protocols: A Guide to Methods and Applications (Innis, 2012). Forward primer 5'-AAACCCACAAGCCCCTCTGA-3' and reverse primer 5'-GATGAATTAGCCCACCCAAAC-3' were found in Mohammadreza Ostadali and colleague's work on dopamine receptor genes (Ostadali, 2004). A mixture of 15 ug purified DNA, 2.0 ug each primer, 2.0 ug DNTP mix, and 0.5 ug of taq polymerase were mixed in a small test tube and placed in a thermal cycler to run 35 cycles of 30s at 95°C denaturation (5 min initial denaturation), 45s at 50°C annealing, and 30s at 68°C extension (5 min final extension) (Peake, 1999). This process was repeated with different purified DNA samples for a total of three trials. The annealing temperature was calculated to be roughly 3-5°C below the lower melting temperature of the two primers (Wu, 2012), which were given by the primer manufacturer.

     Gel Electrophoresis

     Materials:

• 0.5g Agarose Powder
• 17.5mL 20x Lithium Borate (LB) Solution
• 5uL SYBR Dye
• 5uL Gold Bio 100bp Ladder
• 5uL PCR Product
• Deionized (DI) Water

     Agarose powder and 2.5mL of LB solution were mixed in an erlenmeyer flask with 47.5mL DI water to dilute to a 1x LB and 1% agarose by weight solution. The flask was then heated in a microwave to near boil then set aside to cool. 5.0uL of SYBR dye was added and mixed into the solution and the dyed solution was poured into a gel mold with comb and left to set. Once set, the gel was placed into the electrophoresis chamber and the remaining 15mL LB solution was diluted to 1x concentration and poured into the apparatus just enough to cover the gel. 5 uL DNA ladder was then pipetted into the leftmost well and 10 uL of the PCR product into the adjacent well(s) before closing the chamber and running at 170V for 10-15 minutes. The second and third trials were run separately from the first, but under the same conditions. The finalized gels were placed in UV light for better viewing.

     Data Analysis

     Both human and squirrel subjects were filmed on site to be analyzed afterward. Squirrel videos were analyzed by second to be showing one of the following behaviors: Scampering, Foraging, Searching, Burying, Chewing/Eating, Alert, Fleeing, or In Tree to display the relative abundance of these behaviors as observed. These behaviors were elaborations of the behaviors defined by Trevor Poole in Happy animals make good science. These are alertness, business, foraging, relaxed state, and abnormal behavior (Poole, 1997). Videos were split into 30 second intervals in which the subject was assessed to either exhibit or to not exhibit each of these behaviors. These totals were then split by the location the data was collected, displaying similarities and differences in behavior between locations (Partan et al., 2010). To compare the relationship between our dopamine releasing behavior (foraging) and our satiation related response (relaxed state), a Chi square test of independence was performed with variables Foraging/Not Foraging and Relaxed/Not Relaxed.

     Human subject videos were split into pre/during/post smoking action segments and the presence of satiation related facial movements and positions defined by Charles Darwin and discussed by David Matsumoto (Matsumoto et al, 2008). These included: lip protrusion, mouth open, laughing, purposeless movements, nose wrinkled, skin under eyes wrinkled, upper lip raised, partial closure of eyelids, turning away eyes, and mouth drawn back at corners. Number of occurrences of these behaviors was recorded for each interval for each recorded instance of smoking and displayed on a bar graph.

     Gel electrophoresis results were analyzed from a still photo of the gel by recording the number of pixels along the image each band had traveled. Migration distance of each rung of the ladder was plotted with its respective size on a semi-log plot along with an exponential best fit equation. Migration distances of test lane bands were compared to the ladder plot with this equation.

     Final Author: [B-680]

     Fox squirrels showed different frequencies of the same satiated behaviors in urban versus secluded areas. For both rural and urban squirrels, the most abundant behavior was busyness; thirty for urban and twenty-two for secluded squirrels. For alertness, we see sixteen squirrels in the secluded area, and nine squirrels in the urban location. In both the secluded and urban area we observe fifteen squirrels foraging. Thirteen secluded squirrels show the relaxed satiated behavior versus six from the urban squirrels observed, indicating that fox squirrels in secluded areas are more relaxed with their surroundings. Both exhibited abnormal behavior, which included tail flicking, however secluded had more observations than urban (Figure 1B).

     Scampering was considered general movement with frequent stops, foraging was observed as the squirrel searching in the ground or has not found food. Searching was deduced as having a nut and looking for a place to bury it, and fleeing was running away from an observed threat. Burying and searching, which are both indicators of foraging, were the most prevalent, at 20.8% and 24.8%. Most squirrels were observed on the ground, however, 17.3% of squirrels observed displayed these behaviors up in a tree (Figure 2A). A graph was created to analyze the dopamine releasing behaviors in squirrels (Figure 2B) . Foraging and relaxed behaviors were one of the major satiated behaviors observed in squirrels (Poole, 1997). A comparison was made between foraging and relaxing among squirrels. Thirty one squirrels were observed to be foraging, eight squirrels were relaxed and ten were observed to be exhibit both behaviors. A Chi Squared test was performed comparing these two satiated behaviors, and later a t-statistic was calculated giving a p value of 0.5238, which gives very little evidence that foraging and relaxing in squirrels is related to each other.

     Humans appear to exhibit satiated behaviors while smoking and after smoking. The footage was reviewed by a pair of researchers who were trained to recognize satiated behaviors. A total of fourteen pedestrians were observed to be smoking and ten different behaviors were observed. Those behaviors were: lip protrusion, mouth open, laughing, purposeless movements, nose wrinkled, skin under eyes wrinkled, upper lip raised, partial closure of eyelids, turning away eyes, and mouth drawn back at the corners. There were twelve occurrences of satiated behaviors observed during the pre-smoking phase, thirty five were observed while smoking, and sixteen were observed during the post-smoking phase (Figure 3a). Laughing, mouth open, and lip protrusion were frequently observed over other behaviors. Partial closure of eyelids and upper lip raised were only observed in the during phase. Based on the data, a greater number of satiated behaviors were observed from the during and post phases. Evidently, there is a relation between smoking and satiated behavior in humans.

     PCR amplification and gel electrophoresis tests were conducted to find evidence of DRD1 gene in humans. The Gold Bio 100 bp Ladder shows an especially bright 500bp band as seen in well C (Figure 4A). The ladder migration from our first gel is shown (Figure 4B), which was discarded because no band showed up and is not shown. The best fit line is modeled by the equation y = 2442e^-0.0323x. Lane B shows a smear that was likely a result of sample denaturing before amplification (Figure 4A). A bright band appears in lane A near 500 bp, implying the amplification was successful. The equation derived from the first gel image cannot be used to test the validity of our results in the second gel.

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