Finalized By: Allison Vlk

     Midwestern birds migrate to warmer locations during the winter, while others stay put in their natural habitat (Jenni, 2003). For those that stay, aggression levels can increase because of the seasonal temperature variation (Soma, 2006). In general, testosterone levels typically decrease during non-breeding seasons, however, aggressive behaviors are still prominent during these winter months (Soma, 2006). It is important for birds to sustain themselves during nonbreeding seasons and in the colder months in order to have a healthy transition into mating season (Lahti et al, 1998). Therefore, many birds will become combative at feeders in accordance with temperature change and resource availability (Stokes, 1961). If temperature is decreasing due to seasonal variation, then aggressive behaviors in Midwestern birds will become more frequent because altering testosterone levels amplify aggressive behaviors.

     Behavioral differences in birds, particularly aggression, can be linked to specific genes. In birds, aggressive behavior is often linked to a series of genes that regulate testosterone levels (Archer, 1991). In particular, the AR gene is associated with the making of the protein androgen receptors to decipher signals from male hormones such as testosterone in male body tissues (Rosvall , 2012). Testosterone has been found to increase aggressive behavior in numerous vertebrate species, including birds (Archer, 1991). Circulating levels of testosterone are accurate predictors of aggression (Rosvall, 2012). This gene expression of molecules associated with sexual hormones in the brain are linked to aggression (Rosvall, 2012). Additionally, it has been found that sensitivity to testosterone in different individuals can cause aggression regardless of just circulating testosterone levels (Rosvall, 2012). Thus, the differing individual hormone signaling and sensitivity are thought to affect aggression, which accounts for aggression in accordance to low circulating testosterone during non-breeding seasons (Rosvall, 2012). Seasonal variability, food availability and survival stressors, influence the period of time for reproduction in mating seasons, influencing the testosterone levels within individuals (Russ, 2015). Differing hormonal signals and varying testosterone levels, in accordance with seasonal abiotic factors, may influence aggressive behavior of Midwestern birds (Archer, 1991; Rosvall, 2012).

     In comparison with bird aggression, human aggression can also be assessed. When just considering humans, human aggression has been studied and it is believed that aggressive tendencies are more visibly exhibited as resources become more scarce due to climate change (Hsiang et al, 2013). It has been noted that as there has been a subtle global change in temperature with great impacts through past decades. After experimentation, it was found that the outcome of conflict was relatively correlated with climate change (Hsiang et al, 2013). In addition, the global temperature has gradually increased, in which human aggressive tendencies can be directly correlated through past research conducted (Hsiang et al, 2013). Furthermore, it has been documented that colder temperatures have up to a 40 percent increase in aggressive tendencies based on a deviation of temperature changes (Hsiang et al, 2013). Upon thorough examination into human behavior, human aggression heavily revolves around serotonin (Craig and Halton, 2010). Serotonin is a hormone and neurotransmitter that regulates happiness and well being (Craig and Halton, 2010). This means that there must be enough receptors present for serotonin; not enough binding sites can lead to low levels of the hormone, and therefore could lead to disorders, like depression (Craig and Halton, 2010). Furthermore, the gene MAOA (Monoamine Oxidases) encodes and regulates enzymes that break down serotonin and other neurotransmitters (Craig and Halton, 2010). Experiments were conducted that tested the MAOA gene and proved that it was linked to higher aggression levels (McDermott et al, 2009). The MAOA gene also has observed cases of null mutations (mutant copy of gene) that leads to imbalances in serotonin levels, which is a way that human aggression has been shown to be amplified (Craig and Halton, 2010). Our experiment observed this gene in the human's actions while interacting at feeding stations during the winter. It is important to study the MAOA gene because we are able to link human aggressive tendencies to a genetic component, which can also be triggered by the environment, in this case possibly changing temperatures (Craig and Halton, 2010). If temperature is decreasing due to seasonal variation, than aggressive behaviors will become more frequent in humans because altering testosterone levels amplify aggressive behaviors.

     In studying the aggressive behaviors of humans and birds while the temperature is changing, there are numerous conclusions that could be predicted: the aggression, in both birds and humans, will decrease, increase, or not change at all because temperature changes can lead to increased aggressive behaviors as organisms struggle to survive (Stokes, 1961). Duckworth conducted experiments studying how Western Bluebirds' behaviors changed, while protecting their nesting areas, over three breeding seasons. It was found that the birds behaviors were repeatable and showed a relationship between aggression and mating season between species (Duckworth 2006). While pooling together the ideologies from Soma, Hsiang, and Duckworth, we hypothesized that as the temperature decreases as the seasons change, birds and humans will exhibit more aggressive behavior because altering testosterone levels amplify aggressive behaviors. (Duckworth, 2006; Hsiang et al, 2013; Soma, 2006). It is known that in order to survive, animals must maintain and protect their resources as the cold weather hits; winter begins to set in and resources begin to dwindle (Jenni, 2003). Therefore, it is expected that as the seasons change and the temperature varies, the birds will become more aggressive at the feeders. As the season changes from fall to winter, resources that are available for animals to eat rapidly dwindle, and so it is expected that the birds will become more aggressive when observed at the feeders in an attempt to fight for the little food that remains (Jenni, 2003).

Finalized By: Melanie Bumler

     Midwestern birds were observed to determine their aggressive tendencies while interacting at their feeding stations as the temperatures decreased during the season change. One feeding station, the undisturbed feeder, was observed behind 919 East Shaw Lane just off of River Trail in East Lansing Michigan during the study. All data was collected in East Lansing on Michigan State University's campus.

     Aggressive tendencies in birds related to seasonal variation and temperature along with their interactions were observed. Aggressive tendencies were classified as raised wings, beak display, puffed chest, and ruffled feathers. Feeding stations were observed two times a week, at time intervals of 8 am to 9 am on Monday and Friday for eight weeks during the fall semester. The temperature was obtained on the days of observations at the location of the feeder using a scientific outdoor thermometer (measured in degrees celsius). A thermometer was purchased from Scientific Instruments Inc. Interaction between multiple Midwestern birds were observed. All visual observations of aggressive tendencies along with no aggressive behavior were recorded individually with a tally; as one of the behaviors was observed a tally was marked under that behavior. This allowed for a large sample size. Two group members were positioned at an appropriate distance in relation to the location of the feeder in order to eliminate subjectivity when documenting behaviors. These two group members solely observed birds.

      Observations from the field at the undisturbed feeder were recorded and organized. The observed behaviors were sorted into the number of times that the behaviors occurred in accordance to the temperature and the week of the recorded data. Temperatures were grouped into a temperature range, such as 15 degrees Celsius- 17 degrees Celsius. Aggressive behaviors were documented as total occurrences during the observational periods. If one bird was present, the bird was to be observed in general behavior and act as a control.

     Human aggression behavioral cues were studied by observation. This observation took place at the Starbucks on the corner of Grand River Avenue and Charles Street. There was one observation time twice a week, consisting of a length of one hour between 8 and 9 am. During these times, the other two group members were positioned at a table, towards the corner, in Starbucks to record the observations. A group member was also sitting on a bench outside to observe the behaviors that generally occurred on Grand River. This process took place on Monday and Friday each week for 8 weeks throughout the course of the fall semester. Throughout the testing weeks, temperatures were recorded before the group member observed the behaviors at the testing stations. The same thermometer from the bird experiment (recorded in Celsius) was used and recorded before observations for the day began. During the observation days, there were various characteristics that were recorded. Aggressive behaviors were considered as eye rolls, broadened chest, change in vocal pitches (i.e. shouting), and physical contact. These aggressive behaviors were designed to mimic the behaviors chosen by Stokes in his research (Stokes, 1961). These behaviors were measured by counting the amount of times each behavior took place, identical to the bird experiment (Stokes, 1961). If no aggressive behavior was observed, a tally was placed under a column labeled "no aggressive behavior". Temperature ranges were used to group aggressive behaviors seen by the observer. The frequency of each behavior was analyzed at the end of the 8 week period, individually, and as a collective group of aggressive behaviors. Recording and studying human interaction through observing humans on Grand River outside of feeding establishments served as the control group.

      Histograms were used to analyze temperature variation in regards to agonistic behavior. The graphs categorized the temperature at the time of the experiment and the aggressive tendencies of the customers as they ordered their coffee. The behaviors the customers exhibited towards the barista and/or other customers were recorded as well. The data was analyzed using the chi square test of goodness of fit (Franke et al, 2012). This test compared previous research linking temperature and aggressive behaviors by Allen Stokes to our experimental findings (Franke et al, 2012; Stokes, 1961). The degrees of freedom were used to determine the variation of the aggressive behaviors, which was calculated using the n-1 calculation, where n represents the number of variables (Franke et al, 2012). The p value was also calculated to determine statistical significance of the experimental data. If the p value between aggressive tendencies as the temperature drops is less than .05 than the data was considered statistically significant (Stokes, 1961). Standard error was shown by error bars.

      PCR tests were used to amplify the androgen genes present in midwestern birds and humans (Garibyan and Avashia, 2013). Samples of hair, blood, and skin of Midwestern birds were collected from the department of mammalogy and ornithology at Michigan State University (Garibyan and Avashia, 2013). Similar samples were collected for humans at the biology department at Michigan State University. Primers were needed in order to specify the DNA product that was necessary for amplification (Garibyan and Avashia, 2013). These primers were ordered from a biological laboratory company. In order to have assay sensitivity, the presence of template DNA, nucleotides, primers, and DNA polymerase were mixed in vitro (Garibyan and Avashia, 2013). This mixture was placed in a thermocycler which was required to denature and anneal the complementary DNA (Garibyan and Avashia, 2013). The product from the PCR was stained with a chemical dye and gel electrophoresis was used to analyze the product (Garibyan and Avashia, 2013). Gel electrophoresis separated the DNA samples according to their molecular size. The agarose gel was made by measuring out 0.5 grams of agarose and mixing diluted TBE (45 mL of H20 and 5 mL of TBE) with the agarose to make a solution. The solution was then microwaved in a microwavable flask to a light boil. 5 microliters of SYBR dye was added to the solution and left to cool for around 30 minutes. Once the gel was made, equal DNA samples were placed into the wells present in the gel, along with the DNA ladder. The DNA ladder contains known base pairs to compare to the human DNA sample and the bird DNA sample. PCR was used to run samples down their lane; both samples were compared with the results from the DNA ladder and results were analyzed.

Finalized By: Karly Wilson

     We predict that the unaggressive and neutral behavior of Midwestern birds that will be observed will consist of a relaxed demeanor (Figure 1A) with no interaction between birds because it is considered the non-aggressive action within bird species (Stokes,1961). We predict that the aggressive behaviors that will be observed will consist of the puffing or rising of their chest and their wings opening, as depicted in figure 1B and 1C, because they are considered the threatening actions within bird species (Stokes, 1961).

      As the fall season progressed there is no statistical significance to show that Midwestern birds exhibit an increase in agonistic behaviors. At the temperature interval 15-17 degrees Celsius, 20 aggressive behaviors were observed and 23 were to be expected (Figure 2). At the interval 18-20 degrees Celsius, 21 aggressive behaviors were observed and 26 were to be expected (Figure 2). Between 21 and 23 degrees Celsius, 39 aggressive behaviors were observed and 31 were to be expected (Figure 2). At the temperature range of 24-26 degrees Celsius, 35 aggressive behaviors were observed and 35 observations were to be expected (Figure 2). The chi-squared goodness of fit test on Figure 2 produced a p-value of 0.332, which is greater than a p-value of 0.05.

      There is no statistical significance to suggest that humans display more aggressive behaviors as the season progressed. Additionally, during the study period, the temperature did not go below 15 degrees Celsius. Within the temperature range of 15 to 17 degrees Celsius there were 7 observations of aggressive behavior and 6 were to be expected (Figure 3). At the interval marked 18 to 20 degrees Celsius, 10 aggressive behaviors were observed and 7 were to be expected (Figure 3). Between 21 and 23 degrees Celsius, 6 aggressive behaviors were observed and 8 were to be expected (Figure 3). At the interval marked 24 to 26 degrees Celsius, 8 aggressive behaviors were observed and 10 were to be expected (Figure 3). The chi-squared goodness of fit test on Figure 3 produced a p-value of 0.503 which is greater than a p-value of 0.05.

      Because of the observational study conducted by Allen Stokes on the various bird behaviors displayed throughout a seasonal temperature change, we predict that similarly there will be multiple behaviors that humans express to show aggression (Stokes, 1961). Figure 4A includes the various behaviors that are predicted to occur (eye rolling, shouting, broadened chest, physical contact, or no response), and it is predicted that these behaviors will occur more frequently at Starbucks because aggressive tendencies increase at feeding stations (Hsiang, 2013; Stokes, 1961). It is proposed that as each experimental day occurs throughout the semester, each day will have a higher percent of the predicted aggressive behaviors occurring (Figure 4B) because of the seasonal change throughout the experiment (Stokes, 1961).

      We predict that similarities in the base pairs of Midwestern birds and humans will be a result of homology because of the shared evolutionary history between birds and humans (Eisen, 1998). We predict that convergence will infer that Midwestern birds and humans have similar genes that influence aggressive behaviors because of a common ancestral link and the importance of the AR gene through subsequent generations (Eisen, 1998).

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Finalized By: Jared Burge

     Figure 1. Predicted Aggressive Behaviors in Midwestern Birds. Depicted are some of the expected behaviors of bird in the Midwestern regions during temperature change and season progression. A.) Shows what is considered as a nonaggressive behavior of a Midwestern bird. The bird in A is relaxed with no visible outward body language. B.) A Midwestern bird opening its wings, is a behavior that is expected to see as the temperature decreases exhibiting aggression (Stokes, 1961). C.) Portrays a Midwestern bird with a puffed or risen chest, another expected agonistic behavior (Stokes, 1961). The percentage of displayed aggressive behaviors is expected to increase as the season progresses from fall to winter and the temperature decreases (Stokes, 1961).

      Figure 2. Results of the correlation between varying temperatures (degrees Celcius) and the frequency of agonistic behaviors of Midwestern birds. Midwestern birds will be observed twice a week at two feeding stations located at Holmes Hall. Behaviors such as wing raising, beak display, puffed chests, and ruffled feathers were classified as agonistic behaviors for determining the frequency of agonistic behaviors (Stokes, 1961). If these behaviors are present during the observation, the number of these behaviors were to be counted and summed for that day. Temperatures were recorded at the time of observation. A chi-squared goodness of fit test was used to establish any significance in correlation between temperature and the frequency of aggressive behaviors. Expected behaviors were calculated by using proportions of agonistic behaviors during four different trials from Stokes's research in relation to the total agonistic behaviors for those observations (Stokes, 1961). These proportions were multiplied by the frequencies within a given temperature interval, in order to calculate the necessary p-values. The p-value of 0.332 for p<0.05 showed no significance among recorded temperatures and the frequency of Midwestern birds using Stokes's proportions (Stokes, 1961).

      Figure 3. Temperature outside while aggressive behaviors were displayed from each human while at the feeding station. The feeding station was located at the Starbucks on the corner of Grand River Avenue and Charles Street. Observations of aggressive tendencies included eye rolls, shouting, broadened chest, and physical contact which were designed to match similar behaviours to that of the agonistic behaviours of birds in Stokes's research (Stokes, 1961). Temperature was recorded before the beginning of each observation session which is displayed on the x-axis in Celsius. All types of aggressive behaviors were counted and calminated into one single data set which is represented on the y-axis. If no aggressive behaviors are shown by the human a data point will appear at zero.Temperatures on the x-axis proceed from lower temperatures to higher temperatures. A chi-squared goodness of fit test was used in order to identify a significant connection between temperature and the number of aggressive behaviours shown in humans. Expected values were calculated by applying Stokes's agonistic behaviors for each of the four trials he performed and the total number of agonistic behaviors to the human observations (Stokes, 1961). A p-value of 0.503 was calculated for p<0.05, indicating no significance for the temperatures recorded and the correlating frequencies of agonistic behaviours for humans.

      Figure 4. The predicted observations of human aggressive behaviors and the amount that each will occur throughout the experiment. Each graph displayed will display the experimental observations that took place at the Starbucks on Grand river. In addition, the control group which was the observation of human behavior on Grand River will also be displayed. Each will represent the predicted results of human behaviors that display aggressive tendencies. Each behavior was recorded 2 days a week, and at the end of the 8 week experiment, each were counted. The experiment is designed to see if human aggressive behaviors increase at a feeding station, which Starbucks represents (Stokes, 1961). The control observations are represented by the aggressive behaviors observed outside of feeding stations generally on Grand River Ave. In Part A, the x-axis will represent each predicted behavior of aggression, while the y-axis will represent the predicted count of each human behavior, which will be counted using a tally system (Stokes, 1961). The no response will represent that no aggressive behaviors were recorded on an observing day throughout the experiment. Part B will represent the predicted percent of each behavior seen over each experimental day in line graph form. Each of the behaviors represented in Part A will have their own separate trend line for Part B (dashed line, etc.). Part B is predicted to show that as the experiment progresses, the percent of each behavior expressed during each day of the experiment will increase due to changing testosterone levels among individuals as seasonal variation occurs (Stokes, 1961). The x- axis will represent each experimental day, and the y-axis will represent the percent of each behavior.

      Figure 5. Documentary of Observational study showing agonistic behavior of Midwestern birds and humans related to temperature variation.