Responsibility by: Paige Pieczarka

     The fight-or-flight response becomes prominent when exposed to external stimuli among many species. It is a physiological response which occurs when the body senses any threat, danger to survival or any attacks from predators (Jansen, 1995). The fight or flight response was coined by Walter B. Cannon in the 1900s (Jansen, 1995). In other terms the fight or flight response refers to stress or aggression, due to the sympathetic nervous system releasing various neurotransmitters such as adrenal catecholamines from the hypothalamus (Arnaud, 2008). This aggression or stress also leads to a communication among species. Humans and ducks have specific responses when faced with external stimuli. For example, when ducks are faced with outside stimuli they may respond with aggression towards the predator, use alarm calls to signal to ducks to either go away or come help (Gill, 2013). In humans similar behavior occurs when faced with a threat. In many cases they will fight, call for help or run away. Certain behavior and communication become apparent when faced by external stimuli especially among ducks and humans.

     This response occurs in animals when they face various types of danger where the species must fight for survival or flight for their safety. This reaction takes place when the hypothalamus, which is responsible for hormone production, activates the sympathetic nervous system and the adrenal-cortical system (Jansen, 1995). The sympathetic nervous system is a part of the autonomic nervous system and regulates body's unconscious actions. The adrenal cortical system that produces hormones that are released into the bloodstream (Jansen, 1995). The response occurs when neurons fire and the amygdala signals to the hypothalamus to then activate the sympathetic nervous system and adrenal cortical system. Next, the pituitary gland, adrenal medulla, glands and smooth muscles are activated which secretes adrenocorticotropic hormones (Chobanian, 2013). Once, the adrenal gland is activated the production of cortisol as well as the production of adrenal catecholamines including adrenaline and norepinephrine, which is then activated by the adrenal medulla (Chobanian, 2013). Adrenaline is responsible for a surge of energy, while cortisol suppresses the immune system, increases blood pressure and norepinephrine increases response time and heightens focus (Klein, 2016). Other bodily reactions involved in this response is that the respiratory rate increases, blood is redirected from the digestive system to go to muscles and limbs to fight or flight (Neimark, 2016). These systems combine to activate the fight or flight response. The two species that will be compared in our experiment are humans and ducks. The researchers expect that ducks will participate in a greater fight response and humans participate higher in the flight response. Ducks are projected to have increased fight response due to being a wild animal and have a greater instinct to protect against their various predators. The researchers expect more humans to flight than fight in response to the various predations vocalizations due to need to survive that is experienced by the species. The varying data will correlate to the findings predicted due to the hypothalamus being triggered during fight-or-flight response in humans and ducks

     Genes control much of our everyday life and this includes our responses to many situations. For example, the SRY gene is significantly responsible for human behavior. The SRY gene supplies the information necessary for making the protein responsible for the Y protein which is sex-determining. Out of the 46 chromosomes in a human cell there are two allocated for gender, the X and the Y chromosomes (Waters, 2007). If there is XX genotype you develop as a female and if there is a XY genotype you develop as a male. The SRY gene is located on the Y chromosome and that is where the protein processes which causes a fetus to develop testes and not a uterus or fallopian tubes which are the female counter parts to testes (Waters, 2007). This SRY gene in many cases plays a major role in males fight-or-flight response. This male response being in many ways aggression which tends to lead to a fight response. The SRY mRNA is expressed in the hypothalamus, a regulator of the fight-or-flight response (Dewing, 2006). The SRY gene has practical part in the sympathetic nervous system, this being the adrenal medulla which results in an increased blood pressure due to elevated levels of norepinephrine when involved in situations that are stressful or dangerous (Lee, 2012). Also, the SRY gene directly involves the increase of catecholamine which includes adrenaline and norepinephrine being released as well as increased blood flow to muscles and organs in the frontal cortex. This increase activates the aggression, therefore increasing movements in the body geared towards the fight response (Lee, 2012). This activity is lacking in females response due to the absence of a Y chromosome. From this the researchers expect to see increased fight response in males and not females.

     The SRY gene in humans correlates with the SOX9 gene in mallard ducks. Mallard ducks are vertebrates so the mechanism of sex determination is not the exact same for all classes. In many vertebrates the female has the genotype ZW which is a heterogametic sex and the males have a ZZ genotype which is homogametic. The Z chromosome relates to testes development and the W chromosome is needed for development of ovaries (Kent, 1996). The SOX9 gene identifies the male sex by binding to a partner transcription factor and not the DNA site itself. The SOX proteins are transcriptional regulators that mediate DNA binding. Vertebrate genomes contain about twenty SOX members. For sex-determination the SRY activates SOX9 with a specific factor specifically the testis enhancer. After the SOX9 gene has been activated, it is maintained in in sertoli cells which are cells around the spermatids. This goes into the activation of other genes that play a crucial role in testis development such as AMH (Takada, 2005). The SOX9 is a part of the SRY-type gene family; this indicates that their sex-determination factor is related to behavioral responses (Takada, 2005). SOX proteins have been identified in the nervous system. Mostly expressed in the central nervous system, and is essential for neural stem cells (Takada, 2005). The SOX9 protein is crucial in sex-determination, and because of its relation to the SRY family and activity in the central nervous system this gene is assumed to play a role in behavior responses.

     In order to spot the SRY gene in humans and the SOX9 gene in mallard ducks, a PCR machine will be in use to identify these genes. This is a molecular biology method that can identify both DNA and RNA sequences by molecular weight. PCR stands for Polymerase Chain Reaction and was founded by Kary Mullis in 1985. The machine, itself, amplifies many copies of DNA segments. These print-out segments will allow us to classify and analyze where our specific genes are located relative to the other genes expresses on the segment. The PCR copies sections of DNA through a heating cycle. Heats the DNA strands so it splits apart making it easier for primers to bind of the sequences. This heating cycle is repeated and one can get up to thousands of copies of the sequence (Nantel, 2013).

     In this research project the gene analysis and the data from the playback calls will help the research team understand the anti-predator strategies used in mallard ducks and humans. The fight or flight response mechanism in both mallard ducks and humans incorporates communication among potential predators. An anti-predator strategy that is commonly exhibited by ducks is tonic immobility, which is an innate behavioral response that induces a physical immobility similar to catatonic-like state, in order to decrease the attention of the predator response. (Arnaud, 2008). Another reaction that ducks have to the presence of a predator is characterized by hyper excitability and violent escape reactions. All of these are considered a flight response. Signs of the fight response can be recognized as aggressive acts, including biting, threatening, vocalizations, and wing flapping. In regard to humans, many flight responses include: anxiety, poor concentration, and depression. In fight responses humans experience tension in muscles, racing heartbeat, and shallow breathing. Knowing these common behavioral responses to threatening external stimuli in mallard ducks and humans, researchers will be able to identify and analyze communication through fight or flight responses. In conclusion, our hypothesis indicated that, if researchers play predation calls to both mallard ducks and humans, males will respond more with a fight response than females.

     Responsibility by: Reshma Parikh

     In order to gain a better understanding for the fight or flight reaction mechanism within ducks, geese and humans, the team conducted observational experiments using a playback method to stimulate a behavioral response. The team recorded the behavior exhibited by the ducks and categorized these behaviors under fight, flight, or no response. the location of the experiment is on the portion of Red Cedar River that lies behind Wells Hall and the Hannah Administration Building. The team observed the Mallard Ducks that congregate at this location. The team tested to see if different predators' alarm calls cause different behavioral responses in the ducks.

     Playback experiments are often used during observational tests to see if certain audio recordings alter an organisms behavior. The researchers used common predation calls among Mallard Ducks. This included vocalizations from a Great Horned Owl, a Red Tailed Hawk and an American Bald Eagle because they are common predators of Mallard Ducks in the East Lansing area. Also, the team played a chainsaw call as a positive control, because the team expected the ducks to always respond in a "flight" response. Before conducting the experiment, the researchers granted permission to perform a playback experiment on Michigan States campus. When this was granted, the team went out with an iPhone, a Bluetooth speaker and also a notebook to record observations. Each predation call was played using an iPhone that was connected to the Bluetooth speaker. The Bluetooth speaker was used to make sure the sound is loud enough for the organisms to hear. A control speaker that plays no sound was also utilized to make sure the animals were not just attracted to the looks of the speaker. This experiment was conducted over the span of three months. The research team played each predation call for ten minutes and did this for a total of 7 trials to utilize replication and minimize error in the research project. The team also recorded if the organism was a male or a female. This was to see if gender played a role. After each trial, the characteristics of the alarm call and also the behaviors of the ducks and geese was recorded (Manser, 2001).

     We replicated these methods to observe fight or flight responses in humans. We used playback calls to properly observe and record the responses from humans. In the fight behavior we looked for certain behaviors such as walking away and facial expressions that indicated stress or anxiety. For the flight behavior we took notice of confusion, irritation and aggressive behaviors (Jansen, 1995). In order to receive proper filming and observation we performed playback calls. The location we executed our experiment was in the Holmes Hall Cafeteria. We used a Bluetooth speaker and hid it in a very discrete location, so the data would not be skewed and the humans would not notice who was playing the calls. The speakers would play the sounds at random times to make sure that humans would not locate this sound or were not intentionally staying away from the speakers. We played three specific calls: rustling in the bushes, an emergency vehicle noise, and a chainsaw sound. This experiment was conducted for three months. We played predations calls for a total of five minutes and did this for seven trials at different times of the day. We experimented at different times of the day to make sure that our data was not skewed and to minimize error. We conducted many trials to implement replication and to minimize error in the project. After each trial, the characteristics and behavioral responses were filmed and recorded (Manser, 2001). In regard to statistical analysis we decided to use a chi-square table to organize our data. Specifically, we used the chi square test: independence of two attributes. So, we compared fight responses in males compared to fight responses in females, these were the two variables. With calculations, we used the p-value to compare the data and added error bars into our data to show the statistical significance.

     PCR was the technique utilized to analyze both the SOX9 gene in Mallard Ducks and also the SRY gene in humans. Before PCR could be ran, the research team had to specify which primers to use for each gene amplification. For the human SRY gene, the scientists acquired a primer from Bio-Rad, that would specifically amplify the portion of DNA that contains the SRY gene. This primer was called PrimePCR PreAmp for SYBR Green Assay: SRY, Human Reaction (Bio-Rad, 2016). It specifically attached to the HMG-box-family of DNA-binding proteins, which is the area that contains SRY. The primer that was used for the SOX9 gene was called PrimerPCR SYBR Green Assay: SOX9 (Bio-Rad, 2016). This primer was also acquired from Bio-Rad. This primer specifically bound to the section of the SRY gene that contained SOX9 in ducks. The primer sequence for the SRY gene is nine base pairs long and is as follows: 5 ATTTGTTAT 3 (Swiss Institute of Bioinformatics, 2016). The primer sequence for the SOX9 gene is as follows: 5 GGAACTC 3 (NCBI, 2016).

     After the primers were identified, the scientists had to perform DNA purification. This is the process that purifies the DNA prior to PCR (Patella, Thakur, Patel, 2013). DNA was collected in humans by buccal cell extraction. A sterile cytobrush was inserted into a human mouth, and was then swabbed on the inner buccal area. This sample was stored inside of a cryotube and stored at room temperature (Mulot, Stukcer, Clavel, Beaune, Loriot, 2012). DNA was collected in Mallard Ducks by their feathers. The feather was collected from a local Mallard social area in East Lansing, Michigan. After the feather was collected, a 1 cm portion was cut from the end of the feather at the calamus. These samples were kept in envelopes at room temperatures (Bello, Francino, Sanchez, 2001). These samples were then centrifuged individually at 12,000 times the force of gravity for 10 minutes. Then these samples were individually transferred into separate tubes and the DNA was then purified with phenol, chloroform and isoamyl alcohol is a 25:24:1 ratio (Bello et.al. 2001).

     After the DNA was purified, the PCR analysis began. First, a master mix was made for each sample that included 15 microliters PCR buffer, 2.5 microliters of dNTP mix, 103.2 microliters of sterile PCR water and 2.4 microliters of taq polymerase. 2 microliters of purified DNA were added to each tube along with the PCR master mix. This was then placed in the thermocycler at 95 degrees Celsius for 1.15 minutes, then again at 66 degrees Celsius for 1 minute, then again at 72 degrees Celsius for 3 minutes. 20 cycles were conducted to ensure amplification of the DNA sequences. After PCR was done, the team could perform gel electrophoresis to get a visual representation of the DNA. Gel electrophoresis uses agarose and also UV light (Bello et.al 2001). A 1 percent agarose gel was made using agarose and a buffer solution. This mixture was then heated up inside a microwave. After cooled, 2 microliters of the DNA from the PCR was added. The finalized gel mixture for each separate sample (Mallard Duck and Human) was cooled for 30-40 minutes and added to separate wells in the electrophoresis holding chamber (Mecey, White, 2016). Then, the apparatus was turned on and the process took place. A photo of the electrophoresis was taken using a special imaging system that utilized ultraviolet illumination (Kazmin, Edwards, Turner, Larson, Sarkey, 2002). This photo was used to analyze the differences and similarities between the gene found in the duck and the gene found in a human.

     Responsibility by: Sonora Desai

     Considering the observations that have been made, the Mallard Ducks elicit the similar reactions to all three predator calls (Figure 1a). We have tested the Red-Tailed Hawk, the Bald Eagle, the Great Horned Owl and a Mallard Duck call, and all four calls seem to evoke different degrees of urgency of a similar behavior. This correlates with our prediction, which was that we predicted if an external stimuli was presented, than the ducks would respond with different levels of urgency. (Manser, 2001). Our data proves that prediction (Figure 1b). The ducks elongate their body and neck and stand very still, and begin to move their head in an inconsistent bobbing/twitching motion. For each of the three calls, the frequency of the head bobbing/twitching motion varies slightly. We predicted that the more dangerous the predator or situation is perceived to be, the more prominent the response, which was proved in our data (Figure 1a). (Manser, 2001). According to our data, we found that the the bald eagle received the most responses, and the Great-horned Owl received the least amount of responses (Figure 1).

     Fight-or-flight responses were tested among humans using vocalizations. These danger oriented sounds consisted of emergency vehicle sirens, rustling in the bushes, and a chainsaw noise (Figure 2). We predict that human males will have a greater frequency of fight responses than human females because of the presence of the SRY gene in males, while females will have a greater flight response due to the absence of the gene (Lee, 2012). (Figure 2).

     These behaviors were tested throughout various playback experiments, and were observed and recorded (Figure 3a). The research team then tested differences in fight or flight behaviors amongst genders of Mallard Ducks (Figure 3b). We predict that male Mallard Ducks will show more often than not, a fight response, while females will exhibit mostly flight responses due to the SOX9 gene that is present in males. The SOX9 gene is the sex determining factor, and is also known to code for aggression, which is why the fight behavior would be more common in males. (Takada, 2005). (Figure 3).

     In this experiment, PCR and gel electrophoresis was used to compare and contrast segments of DNA from both the Human SRY gene and also the Mallard Duck SOX9 gene. We predict that through gel electrophoresis, the SRY gene and the SOX9 gene will show similar band size and will also appear at similar locations on the gel (Figure 4). This is because gel electrophoresis allows DNA to move down a ladder based on the molecular weight of the DNA. The two genes are in the same family, and therefore the molecular weight of the DNA should be nearly the same. This will evoke the DNA to move down the gel, and end up in nearly the same location. Also, the brightness of the DNA will be nearly identical when shown under ultraviolet illumination (Takada, 2005). (Figure 4).

     Arnaud, I., Mignon-Grasteau, S., Larzul, C., Guy, G., Faure,D. (2008). Behavioural and physiological fear responses in ducks: Genetic cross effects. Animal, 2(10).

     Chobanian, A. V. (2013). Fight or Flight: Adrenaline by Brian B. Hoffman (2013) Harvard University Press Cambridge, MA, USA. The FASEB Journal, 27(9), 3413-3413.

     Jansen, A. S., Nguyen, X. V., Karpitskiy, V., Mettenleiter, T. C., & Loewy, A. D. (1995). Central Command Neurons of the Sympathetic Nervous System: Basis of the Fight-or-Flight Response. Science, 270(5236), 644-646. doi:10.1126/science.270.5236.644

     Klein, S. (n.d.). Adrenaline, Cortisol, Norepinephrine: The Three Major Stress Hormones, Explained. Retrieved September 19, 2016, from http://www.huffingtonpost.com/2013/04/19/adrenaline-cortisol-stress-hormones_n_3112800.html

     Neimark, N. F., M.D. (n.d.). The Fight or Flight Response - 5 Minute Stress Mastery. Retrieved September 20, 2016, from http://www.thebodysoulconnection.com/EducationCenter/fight.html

     Bello, N., Francinco, O., & Sanchez, A. (n.d.). Isolation of genomic DNA from feathers. Isolation of Genomic DNA from Feathers. Retrieved October 18, 2016, from http://vdi.sagepub.com/content/13/2/162.full.pdf

     Waters, P.D., Wallis, M.C., Marshall Graves, J.A. (2007) Mammalian sex--Origin and evolution of the Y chromosome and SRY. Semin Cell Dev Biol. 389-400. Retrieved 2016

     Dewing, P., Chiang, C. W., Sinchak, K., Sim, H., Fergnagut, P. O., & Kelly, S. (2006). Direct regulation of adult brain function by the male-specific factor SRY. 415-420. Retrieved 2016.

     Lee, J. & Harley, V. R. (2012) The male fight-flight response: A result of SRY regulation of catecholamines?. BioEssays, 454-457. Retrieved 2016.

     Takada, S., Ota, J., Kansaku, N., & Yamashita, H. (2005, October 7). Nucleotide sequence and embryonic expression of quail and duck Sox9 genes. Division of Functional Genomics. Retrieved October 18, 2016, from https://www.ncbi.nlm.nih.gov/pubmed/16216246.

     Kent. "A Male-specific Role for SOX9 in Vertebrate Sex Determination." The Compony of Biologist Ltd (1996): n. pag. Web. 19 Oct. 2016.

     Settin. "Result Filters." National Center for Biotechnology Information. U.S. National Library of Medicine, 2008. Web. 19 Oct. 2016.

     White, P., & Mecey, C. (2016). LB145: Cell and Molecular Biology. MI.

     European Bioinformatics InstituteProtein Information ResourceSIB Swiss Institute of Bioinformatics. (2016). Sex-determining region Y protein. Retrieved November 22, 2016, from http://www.uniprot.org/uniprot/Q05066

     SOX9 SRY-box 9 - Gene - NCBI. (n.d.). Retrieved November 22, 2016, from https://www.ncbi.nlm.nih.gov/gene/6662

     Manser, M. B., Seyfarth, R. M., & Cheney, D. L. (2001). Suricate alarm calls signal predator class and urgency. Trends in Cognitive Sciences, 6(2), 55-57. doi:10.1016/s1364-6613(00)01840-4

     Company, B. C. (n.d.). Journals Menu. Retrieved November 22, 2016, from http://nexusacademicpublishers.com/table_contents_detail/4/187/html

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Responsibility by: Kelly Blakemore. Figure 1: Observed Male and Female Response Types to External Stimuli A. This figure shows results of data collected comparing the male and female Mallard Ducks behavioral responses to each of the three predatory calls: Great Horned owl, Red-Tailed hawk, and Bald Eagle. The team also chose to show the data for the Mallard ducks responses to the sound of chainsaws, to which almost every duck responds with retreating. The Chainsaw sound response was treated as a positive control. The other stimuli used was a no call used for a negative control and mallard duck as another stimulus. This data was collected at the Red Cedar River behind Wells Hall. It was collected using playback methods with a small Bluetooth speaker and an iPhone. The graph categorizes their behavioral responses into female attentiveness and retreating, and male attentiveness and retreating, and recorded the average number of responses we observed at each trial. We found that males tended to show attentiveness more than retreating, and females tended to retreat more. Among the various predatory calls, Mallards overall responded most to Bald Eagle calls, second most to the Red- Tailed hawk, and least to the Great Horned owl. The error bars were created by using the chi-squared test for independence which allowed us to compare multiple categories of data.(Arnaud, 2008). B. After analyzing the data from the first graph, a second graph was made to see the amount of times males elicited a response or no response in comparison to females. Overall, males tended to respond to calls more than females did. Error bars were added to both graphs to represent the standard error (SE) of the average number of responses, and were found using the chi square test for independence. (Arnaud, 2008)

Responsibility by: Kelly Blakemore. Figure 2: Fight-or-Flight Response Among Human Males and Females due to External Stimuli. This graph represents the trend based on responses among gender in the human species. The results indicate the role of fight or flight responses that is most prominent in male or female. The bars indicate gender and are placed per the response that was observed. The external stimuli consisted of emergency vehicle sirens, rustling bushes and trees, chainsaw and no call, this is the overall data that was observed. The data was collected while in Holmes Hall cafeteria, and was found using playback experiments. A small Bluetooth speaker was placed in a discreet location and calls were played using an iPhone. Observations were taken in the course pack. It was found men had a higher fight response and women had a greater flight response. Overall males elicited a greater fight response, while females elicited a greater flight response. A few no responses were collected due to the various stimuli that was experienced while doing the experiments that distracted humans. The error bars represent the standard error (SE) of the average responses, and were calculated using the chi-square test of independence, because it allowed us to categorize and compare two different data sets. (Lee, 2012)

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Responsibility by: Kelly Blakemore. Figure 3. Fight-or-Flight Response of Mallard Ducks A. Mallard duck responded to external stimuli observed while swimming in the Red Cedar river near Hannah Administration building and Wells Hall. The male represented showed a quick movement and a slight head bob which indicated aggressive behavior. This type of response was what the research team considered a fight mechanism. This was taken using an iPhone camera, and converted into a GIF using an internet software. B. This represents the average number of fight or flight responses among ducks observed through series of playback experiments including predator calls and other stimuli. The external stimuli consisted of Great Horned Owl, Red-Tailed Hawk, and Bald Eagle, which are predators of the Mallard duck. Other stimuli consisted of chainsaw, Mallard duck and no call used as a negative control. The data showed that male Mallard ducks are more likely to respond with a fight response, and the female ducks are more likely to respond with a flight response. A few times, the ducks did not respond to the calls. This could be due to other humans around the Red Cedar River, which caused the ducks to be distracted. All of this data was collected at the Red Cedar using a Bluetooth speaker and an iPhone, which was considered a playback experiment. Error bars represent the standard error (SE) of the average number of responses, and using the chi square test of independence which allowed us to use categorical data and compare two data sets. (Takada, 2005)

Responsibility by: Kelly Blakemore. Figure 4. Predicted gel electrophoresis of SRY gene from ultraviolet illumination. A representation of a predicted gel electrophoresis photograph of the SRY gene. The left lane displays the DNA ladder, or the control, which is compared to the DNA that was actually tested. This photograph was taken after gel electrophoresis was ran, and the gel was placed under ultraviolet illumination. Typically, these bands would be very bright in color, but because it is a predicted figure, the bands are shown in a light grey color. They are differentiated, because the slightly darker grey bands would show a brighter band on the photograph. The letters "bp" on the left side of the photograph describe the "base pairs" or band size of the DNA that is shown. Gel electrophoresis works by the DNA moving to a certain point based on the molecular weight. This image shows how SRY would look if the research team ran a gel on that specific gene. (Company, 2016).

Responsibility by: Kelly Blakemore. Figure 5: Documentary summarizing semester research project on Mallard Ducks and Humans. An online documentary describing and showing the semester long research project conducted in LB144. This video was taken using an iPhone and edited using iMovie on a MacBook air.