Communication behaviors between both ducks and humans have long been observed. However, it is rare that the behaviors are compared. The purpose of this experiment was to find and log a communication behavior in Mallard and American Black ducks and discover a homologous behavior in humans. In addition, an attempt to find a corresponding gene was made. It was hypothesized that if Mallard and American Black Ducks were exposed to predation risk using playback sounds, then behaviors exhibited will be similar to reactions in humans because of the abundant presence of the PNMT gene in both species (Steeves et al. 1987). The design of the project was an observational method, where experimenters observed test subjects with negative controls (observation) and positive controls (playbacks associated with predation). Polymerase Chain Reaction was used to amplify the target DNA studied. Andrendic Receptors (AR), such as Phenylethanolamine N-methyltransferase (PNMT), can be observed in virtually all mammals, such as the central and peripheral nervous systems in humans (Goldstein et. al 1972). This is significant, as adrenergic receptors are targeted for many medications (Hoehe et. al 1992). An advanced understanding of adrenergic receptors could lead to quick-performing and cutting-edge clinical drugs. The results show similar behaviors exist between ducks and humans when posed to a predatory risks. Both species reacted to unfamiliar/threatening circumstances. They were either alert and exhibited various behaviors or were motionless with no reaction to the potential risk. The findings were categorized into different response levels and the reactions expressed. PCR experimentation supported this finding because a gene was used to connect the behaviors in both humans and ducks.

     Each kind of animal has a different adaptation as to how to defend themselves against predators. North America is home to a vast amount of animals, such as: 457 mammals, 914 birds, 662 reptiles, over 300 amphibians, and 4,000 known arachnids (Karuga 2017). Since there are so many species, competition for survival is fierce. Adaptations are developed through evolution and different mechanisms have been studied throughout the years, examples being: fear and stress, vocal communication, and camouflage. The research for this study was collected in East Lansing, Michigan, all within Michigan State’s Campus (5,200 acre campus). The most common duck species found on Michigan State's Campus are the American Black Duck and Mallard ducks. The American Black duck tends to prefer the eastern coast of the United States, particularly northeast in the summer and mid to southern eastern during winter and breeding season (Longcore et al. 2000). The American Black Duck is known for totally abandoning an area of predatory risk when they feel threatened (Bowman & Longcore 1989). This is in contrast to a study of white-tailed deer, who were exposed to coyote predation risk, in which, it was found that predator existence did not influence the amount of foraging by white-tailed deer (Gulsby et al. 2018). The other species of duck selected is the Mallard Duck. The Mallard duck prefers calm, freshwater sanctuaries and can often be found throughout Asia, North America, and Europe (Bengtsson et al. 2014). Mallard Ducks have been reported to realize that avoidance of one predator may lead to increased risk of another predator (Englund et al. 2012).
     Communication is an essential factor for organisms growth and survival, as it tests an organism's strength in alertness and reaction. To broaden knowledge on communication, it is important to understand how organisms communicate and respond, especially when threatened with predation. Communication ranges from gaining attention in order to mate to warning each other about possible threats. Organisms such as frogs for example, have a wide variety of communication skills. With the use of sound, frogs use mating calls in groups to find female frogs. The communication is very complex, since in order to find a women, the males with the most attractive mating calls are the most successful (Ryan et al. 1985). This form of communication is vocalization and auditory which essential to learn about to understand the variation of interaction. Not only is communication based around mating, but also on possible predation on a species. Research on Campbell monkeys located in the Ivory Coast found that the monkeys knew about six sounds used to transmit information (Yirka et al. 2015). The unique sounds help warn the groups of species about nearby danger, ranging from slight to major threat. This study not only explains the many forms of communication, but also why it is necessary to be understood. These tests help guide the importance of communication and how organisms, such as ducks, can be tested in a similar fashion. To test the importance of communication and predation among Mallard and American Black ducks, they were observed in a natural habitat. Ducks often show physical communications. For instance, they may nod their head away as a sign of disinterest versus an extended neck showing interest (Inglis et al. 2000). In addition, Mallard Ducks physically express fear and stress regarding predation. When threatened, Mallard Ducks show either vigilance, calmness or preening (Zimmer et al. 2011). By using this information, the question: How do Mallard Ducks and Black Ducks Communicate and Respond when Threatened with Predatory Behavior? can be tested and compared to human genes.
     Both humans and ducks visually as well as auditorily communicate and express fear or stress when a stranger/predator is around. Humans have cognition similar to Mallard and American Black ducks which enables experimenters to test how alike their daily activities may be. Ducklings recognize and gain trust in the duckling-mother relationship and humans have an equivalent bond with their progeny. In fact, if a duckling is separated from their parent post birth, it is quite possible that they grow up following whatever/whoever they first see in the initial stages (Senft, 2017). This applies to children as well when parental attachment is essential to their growth. Securely attached children readily seek out their caregivers when in high stress, but feel sufficiently safe to explore their environment at times of low stress (Moretti, 2004). Fear/stress response in ducks when a predator is around, compared to strangers for children under the age of 6 who may pose as threats or even someone they do not usually interact with, is being tested. This applies to ducks as well since predation may cause them to take action of either fleeting or fighting the opposer off. Epinephrine is the fight-or-flight hormone because it is released in large quantities during times of intense fear or stress in species (Helix 2018). Both humans and ducks secrete this hormone every time they try to respond to a stranger. Phenylethanolamine N-methyltransferase (PNMT) is an enzyme that is primarily found in the adrenal medulla that converts norepinephrine(noradrenaline) to epinephrine (adrenaline) (Goldstein 1972). This gene is found in both humans and ducks contributes to converting epinephrine, the hormone involved in fear/stress responses of both species.It is located on chromosome 17q21-22, with the base pairs AATAAA (Hoehe et al. 1992). Observational studies of both ducks and children (under the age of 6) will determine how effectively these genes work and how much secretion of epinephrine will take place when both species are approached by a foreign organism.
     In order to understand if these behaviors are truly genetically linked, it will be necessary to perform several tests. There are many control factors that will contribute towards gaining data in the experiment which include: observations will only take place on the Michigan State University campus, there will always be at least two one-hour observations recorded in a single week, and the observation will be based on only two species of ducks: the Mallard (Anas Platyrhynchos) and the American Black Duck (Anas Rubripes). Similarly to the work of Zimmer, Boos, Bertrand, and Robin, there will be an increased predation risk when there is an increase in disturbance (Zimmer et al. 2011). This increased predation risk will be based on the fact that prey have evolved antipredator responses to generalized threatening stimuli, such as loud noises and rapidly approaching objects or species (Frid and Dill 2002). Therefore, the experiment will also use disturbance noises: for these noises, a phone will be used to send an auditory signal of a hawk and eagle noise, as hawks and eagles are common predators of these species (Washburn 2018). Duck behaviors will first be defined as either anti-predatory behavior or no response. The first category of anti-predatory behavior will include: vigilance, diving, and running on water (L. Dessborn et al. 2012). They will then be further designated into categories of either alert or motionless. These categories will assist experimenters to further clarify how the experiment will specifically interpret ducks behavior so that both researchers and audience are not confused. Any further actions made by the ducks will be documented in a lab notebook and kept for review. In addition, observations of attachment between children and parents will also take place. According to John Bowlby, in order for a child to survive physically and psychically attachment behaviors must exist and be reciprocated (1958). Attachment behaviors can consist of: tears, tantrums, or even screaming (Solter 2000). This behavior is comparable to the attachment that ducks have to one another. Ducks have social attachments and communicate in similar manners, for example: aggression, incitement, biting, grunting (Darbouze et al. ND). By proposing these methods of study, the anticipation are result patterns between both young children and duck social behaviors that bear similar data; if Mallard and American Black Ducks exhibit predatory responses from the auditory data, then it will be similar to the behaviors that will be noticed in young human children, who view themselves to be at risk, as well.

      In order to obtain data about Mallard and American Black Duck behavior observations were taken along the Red Cedar River in East Lansing and three separate locations were used to have different interactions with the ducks (Figure 1 Left). In location A the researcher (black star) was physically in the water while in location B the researcher was on land and in location C the researcher was on the bridge. In the first figure, the ducks’ initial positions were recorded (in water and on land). In the second figure the ducks’ positions were recorded after the playback sounds of the predator was played on the JBL speaker. The ducks followed their group when posed to danger because they will recognize the security they will have with the same species of their kind (Senft 2017). In situation A, ducks 1-5 swam away being alert but not too reactive. In situation B ducks 6, 7, 8 ran into the water to ‘swim’ away from the potential predator and yet no species was in sight similar to situation A. This is because the volume was too loud for a predator to not be seen in the vicinity. However in situation C, since the researcher was further away from the species, on the bridge, the ducks alerted each other about a predator coming their way, which is why ducks 11 and 13 swam real quick to the other side of the bridge and ducks 9, 10, 12, 14, and 15 flew away from the test site (Figure 1 Right). The figure was modeled after Cavallini’s paper titled Ranging behaviour of the Cape grey mongoose. The home ranges of the Cape grey mongoose in the West Coast National Park in South Africa were recorded in their experiment while duck travel ranges in response to predatory audio was mapped out in this experiment. The experimenters also numbered their animal locations (P. Cavallini 1990) to gain accurate results of each duck at the test location.

Observational Results of Mallard and American Black Ducks

      There were 138 observations obtained for the Mallard and American Black Ducks in this study. Head Turning was a very common reaction displayed by both species (Mallard Ducks=14 observations, American Black Ducks=17 observations), proving that when the call was heard both species were cautious of their surroundings. However, when comparing this to standing still, Mallard Ducks did this reaction more than the American Black Ducks (Mallard Ducks=12 observations, American Black Ducks=8 observations). Although this could be due to sample size, it could be inferred that Mallard ducks tend to stand still when a predator is around, possibly to not draw attention to themselves. On the contrary, American Black ducks head turn and elongate their necks more than Mallard Ducks, demonstrating how the American Black ducks prefer to show vigilance and like to be prepared for the predator. American Black ducks also stood on the water and flew away more, possibly justifying American Black Duck's need to be more active than the Mallard Duck (Mallard Ducks=5 observations, American Black Ducks= 9 observations) (Figure 2). Besides the minor differences, both groups of species displayed similar reactions when faced with a predator call, ranging from head turning to flying away. The observations showed that an auditory call can induce some type of disturbance in the ducks causing a behavioral reaction to occur.

Children Behavioral Patterns Based on Disturbance

     There were fifty seven observations of individuals taken during this study. The five most prevalent behaviors of these studies in order of prevalence, along with a control bar of ‘no response’, were: left the area, head turned, laying down, crying, no response, and yelling. Leaving the area was the highest recorded behavior to a disturbance with a final tally of twelve separate occurrences (Figure 3). The no response control bar, which was classified as ‘no observable response’ ended with a steady number of ten. It was found that the amount of crying instances increased over the remainder of this study, because most instances of disturbance to children included either anger or sadness, which are the two major emotions involved in crying (Green et al. 2011). The behavioral reactions crying, left area and yelling are considered the most intense reaction level, however the observations are lower for those three reactions. This could infer that the children react in less intense reactions compared to no response (n=10) and left area (n=12).

Final Data Analysis

     While observing the ducks to collect our data most of the population studied produced some sort of response when faced with possible predation (Figure 4). The graph was influenced by Figure 1 of Cedric Zimmers research paper, Behavioral Adjustment in Response to Increased Predation Risk: A Study in Three Duck Species. In the figure Zimmer categorizes different stages of the ducks responses and shows the Male Vigilance time for each stage. The response levels were categorized by a high response level, medium response level, low response level, and no response. The percentage of the amount of ducks displaying each level of response was calculated and is displayed using a pie chart. Behaviors that were considered a high response level included standing on water and flying away/diving into water. The behaviors that were considered a medium response level were standing still, grouping together, and communicating with one another. A low response level was considered to be head turning and elongated necks. The highest amount of ducks responded with a low response level (42.11%), so it can be hypothesized that the ducks were aware of the possible predator, however, did not find it to be particularly threatening. The percentage of ducks decreases as the response level becomes more urgent (29.32% of duck responded with a medium response level and 17.29% of ducks responded with a high response level), however, the no response was the lowest (11.28% of ducks) which means we can conclude that a majority of the ducks found the Red Tailed Hawk and American Bald Eagle noises to be a possible risk. The children data was also split up in no response, low response, medium response and high response. No response was the control, low response was head turning and laying down, medium response was leaving the area and high response was crying or yelling. It was found that the highest percent was also low response level (33.2%), which could relate to the ducks since the children reacted in very similar ways such as head turning. Correspondingly, no response was the lowest in children as well, demonstrating how children are also cautious of their surroundings and will react even if the response is low. Figure 4 presents a side by side comparison of the duck and children response levels and shows how identical both species respond to a risk.

PCR Analysis

      The gene that is involved in this reaction is called Phenylethanolamine N-methyltransferase, or PNMT. It converts norepinephrine to epinephrine, the hormones that are involved with fear and stress responses in species. The gene is located on chromosome 17q21-22, with the base pairs AATAAA . The estimated base pair length was around 1886 base pairs for the target sequence. On the right of the PCR figure is the golden ladder, with each band representing 100 base pairs. The top of the figure shows the 3 Wells, the right most well has the 100 BP ladder. Well 2 and well 3 both have the PCR cocktail, Well 2 contains the 3 µl and 5 µl cocktails. Although the target sequence was projected to be 1886 base pairs, the figure does not reach this amount. This could be due to the methodology, specifically the thermocycler steps. The denature and annealing steps of the thermocycler could be altered. This is because the primers were not able to stick onto the DNA sequence. The well contains 23.1 BP which is about the size of the primers, which means the primers did not stick onto the DNA sequence.

Finalized By: B-117

Figure 1: Mallard and American Black Duck positions before and after playing playback sounds. In the figures above, the red circles represent American Black Ducks, the yellow circles represent Mallard Ducks, and the black stars represent the locations of researchers at the times of observations. Spartan stadium, along with Wells Hall and the Bridge behind Wells Hall, are all displayed as well. The scenario on the left represents when the researcher was in the water playing playback sounds (approx. 3 meters away from ducks). In comparison, the scenario on the right represents when playback sounds were played from the land while ducks 6, 7, 8 were on land at the time. Letter C on the graph represents when playback noises were played from the bridge overlooking the Red Cedar River by Wells hall. Ducks 1- 5 in scenario A swam away, while ducks 6-8 ran into the water from the land when predator noises were played. In scenario C, ducks 11 & 13 swam away to the other side of the bridge while ducks 9, 10, 12, 14, 15 flew away from the location. In summary, the methods of the project are pictured, along with an example scenario of what one group of ducks reactions were.

Finalized By: B-117

Figure 2: Children, Mallard, and American Black Duck behavioral reactions when threatening situations arose. (Left) The Mallard duck is represented in green and the American Black duck is represented in orange. Different responses were given between the two types of species. Both species were played a predator auditory cue of a Red Tailed hawk and American Bald Eagle to induce a disturbance causing a behavioral reaction. The multiple reactions were observed and recorded between both species. The most common reactions between both species was found to be Head Turning with a tally of seventeen instances from the American Black duck and fourteen instances from the Mallard ducks. In contrast, the least exhibited behavior for both species was standing on water with tallies of seven instances from the American Black duck and four instances from the Mallard ducks. (Right) For approximately nine weeks, children were observed in a local school, as well as their homes, to determine their innate responses to stress. No stress was purposely induced, rather, researchers waited for stressful scenarios to naturally occur. This included instances, such as: homework, strangers, and not getting what they want. There were fifty three observational participants in this study (n=53). No participant was observed more than once, yet if more than one of the top five reactions were exhibited during their one observance, then it was counted toward each behavior. The top five most common behaviors are represented. No Response is also included as a control. The most prevalent behavior exhibited by children was 'left area' with a tally of the behavior exhibited twelve times. No response, the control, was close behind and tied for second with a count of ten observed instances.

Finalized By: B-117

Figure 3: This figure displays the responses between American Black duck and Mallard duck reactions to predation risk compared to human response reactions to riskThe figure on the left shows the percentage of ducks responding to the Red Tailed Hawk and American Bald Eagle noises with a high response level, medium response level, low response level or no response. Each level was classified with different behaviors that were, based on research, deemed to be high (17.3%) (flying away, diving, standing on water), medium (29.3%) (standing still, grouping together, and communicating with one another), or low (42.1%) (head turning and elongated necks). No response was also included as a control- 11.3%. Children's reactions were also classified based on low, medium, high or no response. Low, which consisted of head-turning or laying down, ended with a percentage of 33.2%. Medium responses (21.1%) consisted of leaving the area. High responses of crying or yelling occurred 28.1% of the time. No response was again included as a control and ended with 17.6% of responses.

Finalized By: B-117

Figure 4: This figure shows the PCR results. The red arrow (left) shows where the estimated gene length would have been (1886 BP). The arrow on the right displays where the band actually ended after gel electrophoresis. The gene that was being searched for is known as Phenylethanolamine N-methyltransferase, or PNMT. It converts norepinephrine to epinephrine, the hormones that are involved with fear and stress responses in species. The gene is located on chromosome 17q21-22, with the base pairs AATAAA . The estimated base pair length was around 1886 base pairs for the target sequence. On the right of the PCR figure is the golden ladder, with each band representing 100 base pairs. The top of the figure shows the 3 Wells, the right most well has the 100 BP ladder. Well 2 and well 3 both have the PCR cocktail, Well 2 contains the 3 µl and 5 µl cocktails. Although the target sequence was projected to be 1886 base pairs, the figure does not reach this amount. This could be due to the methodology, specifically the thermocycler steps. The denature and annealing steps of the thermocycler could be faulty. This is because the primers were not able to stick onto the DNA sequence. Pixel Pro Art was utilized to determine the exact sizing of the pictured band in Well 2. The well contains approximately 23.1 BP which is about the size of the primers, which means the primers did not stick onto the DNA sequence.