Written By: Abigail Miller

Revised By: Abigail Miller

       Different species of birds have a variety of complex characteristics when it comes to methods of communicating and emulating anger. Communication can be as simple as short sounds or long songs (Searcy et al, 2006). Demonstrations of aggression include forceful pecking, kicking, and erect feathers (Dennis et al, 2013). The presence of an intruder can induce any combination of these displays (Dennis et al, 2013). Aggression can be evoked by other species encroaching on nesting territory, limited resources, or the threat of a predator (Arnold, 2000). When one species of bird is not strong enough to drive out the predator they will give off urgency calls to the surrounding bird species for backup (Rasmussen, 2016). This formation of mixed-bird flocks against a predator is known as mobbing (Rasmussen, 2016). Mobbing is a combined form of aggressive displays used to drive a predator out of the bird's territory (Arnold, 2000). We hypothesize that if mixed bird flocks are presented with predator or urgency calls then they will respond with mobbing, aggressive group displays because the flock is collectively stronger than an individual bird.

       Fenner Nature Center which resides in Ingham County, Lansing is home to many species of birds include nuthatches, chickadees, and titmice (Rasmussen, 2016). The eastern screech-owl is common in Ingham County and is a known predator of the bird species stated previously (Nolan and Lucas, 2009). These species of birds exhibit similar behavioral pattern when threatened by a predator, specifically the eastern screech-owl, which allows them to be categorized as a mixed-bird flock (Nolan and Lucas, 2009). Birds join together to perform a mobbing technique because they are not strong enough to eliminate the predator by themselves (Rasmussen, 2016). A mixed flock will form when the birds have a common interest in removing threat, so they will team up to drive the predator away (Farine et al, 2012). The shared benefits from mobbing as a group provide a great mutualistic relationship between the different species of birds (Farine et al, 2012). Due to all the benefits associated with mobbing the mixed bird flocks have been known to attack predators even when they are not an immediate threat which brings some risk to the group mobbing technique (Griesser, 2009). However, any cost associated with mixed- flock mobbing does not compare to the benefits the birds receive from their mutualistic partnership (Farine et al, 2012). These benefits does include optimum security which is conducive to pack type mentality (Farine et al, 2012). If a bird was of and on its own it is more likely to be targeted by the Eastern Screech Owl (Farine et al, 2012).

       Mixed bird species often live together in groups creating a complex alarm system when a predator approaches (Nolan and Lucas, 2009). Signals usually consist of an alarm call then a mobbing call, ranging from high to low frequency, respectively (Bartmess-LeVasseur et al., 2010). However, titmice and chickadees have a system that combine both the alarm and mobbing call into a single signal (Bartmess-LeVasseur et al, 2010). The communication system between the mixed bird species has been identified as a nuclear-satellite system (Nolan and Lucas, 2009). The nuclear species is designated as the leaders; these birds send out the initial alarm calls which will move down to the satellite species (Nolan and Lucas, 2009). Focusing on specific bird species, the chickadee and titmice are considered the nuclear species and the nuthatches form the satellite species (Nolan and Lucas, 2009). Alarm calls from the chickadee and titmice signal to the nuthatches the risk of the predator and level of urgency (Griesser, 2009). Satellite members then respond to the nuclear calls by stopping movement and generating vocalizations (Nolan and Lucas, 2009). Vocalizations continue until the nuclear species signals that the predator is no longer a threat (Nolan and Lucas, 2009).

       The aggressive, territorial mobbing behavior of human males to a predator was previously found to be similar to the mixed bird species response to a screech owl (Calsyn, 1976; Lucas and Nolan, 2009). The 5-Hydroxytryptamine (5-HT) receptor is responsible for the binding of the neurotransmitter serotonin and the 5-HT system regulates the state of aggression (Nelson, 2001; Olivier, 2004). Aggression in humans has been linked to low serotonin levels, causing a greater strain on the cerebral cortex to control behavioral responses that elicit aggression (Crockett, 2011). Birds also have 5-HT receptors for serotonin that have been linked with behavioral changes when the levels fluctuate (Aprison, 1962). The hyperstriatum, a structure only common in birds, becomes stressed when the levels of serotonin are low which stimulates behavioral changes in the bird (Aprison, 1962). The hyperstriatum is comparable to the cerebral cortex in humans, and both these regions of the brain produce behavioral changes when the levels of 5-HT are above or below normal (Aprison, 1962). When the organism is presented with a predator the 5-HT levels fluctuate causing a change in behavior (Manuck et al, 2006). We hypothesize that if human fraternity males are presented with a non member, male, predator then they will respond with mobbing, aggressive group displays, because multiple members are stronger than just an individual.

Written By: Calley Casey

Revised By: Calley Casey

       Observations were taken at Fenner Nature Center located in Lansing, MI south of Michigan State University campus. Permission was obtained by the park rangers of Fenner Nature Center to examine the birds. The behaviors of birds were recorded in a lab notebook during September and October of 2016 during Saturday mornings, 8:00 a.m.-10:00 a.m. The behaviors that were observed were classified in three categories; aggressive pecking, looking, and erect feathers (Dennis, 2013). During observations, there were no threats such as a predator, present. The three behaviors were observed with the use of binoculars in the back of the nature center, past the pond and through a wooded area. When each of these behaviors were observed, a quantitative analysis of the bird's behavior was recorded by marking it down within a chart in the lab notebook. Other behaviors such as, how birds interact with each other and how they act when in isolation, were documented in the lab notebook (Leavesley and Margarth, 2005).

Playback of Eastern Screech-Owl Trills

       A playback method was used in order to observe the aggressive behavior of mobbing that birds tend to show when encountered with a predator. The playback study was conducted after observations. A bose bluetooth speaker was placed on a branch that was at least 1 meter above ground level (Nolan and Lucas, 2009). At the beginning of each trial, the speaker was playing no sound for 14 minutes to document the birds behaviors to serve as a control (Nolan and Lucas, 2009). Along with the behavior, the number of birds that approached the speaker was counted. The screech owl playback file, Megascops asio.mp3, used was from xeno-canto.org. The owl playback was played from an iPhone 5s using the voice memo app and both the iPhone 5s and bose bluetooth speaker were at full volume. The screeching owl playback was played for 14 minutes and observations were recorded (Nolan and Lucas, 2009). Whenever the mobbing action was observed, a quantitative analysis of the birds was recorded by documenting the behavior and the distance between the owl playback and birds within a chart in the lab notebook. Position was categorized into different intervals including 0-2 meters, 2-3 meters, 3-5 meters, 5-6 meters and greater than 6 meters from the bose bluetooth speaker. For each meter increment, twine was used to measure the area in which the birds resided in (Nolan and Lucas, 2009).

Playback of Urgency Calls

       A recording of titmice, nuthatches, and chickadees' urgency call was used from the xeno-canto.org to elicit communication between bird species in the presence of a predator (Nolan and Lucas, 2009). The playback urgency call was broadcasted over the bose bluetooth speaker to replicate the nuclear bird that notices the predator, looking for the satellite bird's response (Nolan and Lucas, 2009).The experiment was performed at 8:00 a.m.-10:00 a.m. and in the same location as the screeching owl playback experiment (Nolan and Lucas, 2009). The bose bluetooth speaker was placed in a tree branch 1 meter above the ground (Nolan and Lucas, 2009). The speaker was broadcasting no sound for 14 minutes and the birds behaviors were documented as a control (Nolan and Lucas, 2009). The urgency call was played from the voice memo app on an iPhone 5s, both the phone and speaker were played at full volume. While playing the urgency call for 14 minutes, aggressive behaviors and distance the bird was from the speaker were documented in the lab notebook as completed in the previous study. The number of call backs to the speaker from the birds were also written down in the lab notebook. Hearing the calls from the birds to the speaker demonstrates the satellite and nuclear species are communicating about the predator in the area (Nolan and Lucas, 2009).

Social Observation

       Over the course of a month an observational study was carried out on Friday and Saturday nights at 11:00 p.m.-12:00 p.m. at a local fraternity house on the Michigan State University Campus. The chosen fraternity house was determined by an online generator, www.randomizer.org. All fraternity member names were taken out of recordings so that their identities were concealed. The identities of the fraternity members and the house were not disclosed in order to maintain the integrity of the organization. To serve as the control, the group members observed the conversation between two fraternity members as one member allowed easy passage of the other member into the house. The "predator" was a non-member male and carried an iPhone 5s, recording the altercation with the voice memo app. The predator started recording as soon as he stepped onto their property. Recording was stopped when a physical altercation took place or if predator felt unsafe. Across all trials, the conversation between the fraternity member and non-member male lasted about 5 minutes. The number of fraternity members that joined in on the mobbing of the predator and the duration of the mobbing were documented in the lab notebook by the other members of the group. Any communication between the members of the fraternity was recorded for comparison to the urgency call playback experiment.

Human PCR

        Polymerase Chain Reaction (PCR) allows for the amplification and duplication of a specific DNA sequence (Garibyan and Avashia, 2013). Specifically, quantitative real-time PCR (qRT-PCR) detects how much of an individual gene appears in the sample (Garibyan and Avashia, 2013). Human DNA was obtained from saliva using a cotton swab and then was combined with a reaction buffer, dNTPs, reverse and forward primers, MgCl2, Taq DNA Polymerase, and PCR sterile water (Lawyer et al., 1993). A PCR kit was purchased from QIAGEN which contained all necessary materials to perform the qRT-PCR test. The mixture was distributed among well plates and placed into a thermal cycler. Results were later compared to the DNA ladder. The machine denatured the selected DNA strands by raising the temperature of the thermal block to 95 C for 30 seconds (Garibyan and Avashia, 2013; Lawyer et al., 1993). The temperature was then lowered back down to 62 C for 30 seconds in order for the primers to attach to the specific DNA fragment (Garibyan and Avashia, 2013; Lawyer et al., 1993). Finally, DNA polymerase attached nucleotides to the new DNA strand once temperature was increased again to 72 C for 30 seconds (Garibyan and Avashia, 2013; Lawyer et al., 1993). This process was continued until 30 cycles had been completed (Lawyer et al., 1993). Final extension of the DNA lasted for 5 minutes at 68 C (Lawyer et al., 1993). After copies of the DNA sample had been completed, the copies were held at 4 C (Garibyan and Avashia, 2013; Lawyer et al., 1993).

Chickadee DNA purification and PCR

       Chickadee feathers were obtained at the Michigan State University Avian Disease and Oncology Department, located off of Mt. Hope Rd in Ingham County. A 0.5 to 1 cm section was removed from the end of the feather quill and placed in a test tube containing 500 ml of lysis buffer and proteinase K at a final concentration of 175 mg/ml (Bello et al, 2003). After sitting for 4 hours at 56 C the feathers were then placed in a centrifuged for 10 minutes (Bello et al, 2003). The contents from the centrifuge were transferred to clean tubes to be purified in phenol, chloroform, isoamyl alcohol, for maximum recovery of DNA this process was done at -20 C overnight (Bello et al, 2003). Master mix was generated from the PCR kit obtained from QIAGEN online shop. This master mix contains a buffer, dNTPs, MgCl2, PCR sterile water and Taq DNA polymerase (Qiagen, Valencia, CA). The master mix was combined in a 12:1 ratio with the purified DNA then sent through the thermocyler then stored at -20 C (Emanual et al, 2013).

Gel Electrophoresis and DNA quantification

       Using the gel electrophoresis kit purchased on the QIAGEN online shop, 1 g of agarose powder and 100 mL of 1X TBE were combined into a 600 mL beaker. The mixture was microwaved for 1 minute in 25 second intervals in order to dissolve the agarose. After, it was cooled for 5 minutes, 3 microliters of SYBR-Safe was added. The agarose gel solution was poured slowly into a 120 mL tank and set for 15 minutes (Emanual et al, 2013). The gel solution was then completely saturated in electrophoresis buffer to approximately 0.5 cm (Emanual et al, 2013). Each well was made by the comb before the agarose set, and was then filled with 5 microliters each of the Human DNA sample, Chickadee DNA sample, and DNA ladder from the thermocyler along with loading buffer (Emanual et al, 2013). The DNA ladder was used to compare to the Human and Chickadee DNA samples. The wells were run at 100 V until the dye runs at least half way down the gel. A spectrophotometer then used UV light to quantify the data. (Emanual et al, 2013).

Written By: Manpreet Kochar

Revised by: Manpreet Kochar

Observations

       Observations began at 8:00 a.m. and lasted until 10:00 a.m. at Fenner Nature Center. A densely wooded area along an unmarked trail contained a large population of mixed bird species. There was a high level of activity early in the morning around 8:00 a.m. While observing there was a wide range of different bird activities. Normal activity consisted of birds flying in groups, birds socializing, and foraging for food. A large group of birds also produced loud vocalizations throughout the two hours of observations. Preliminary data of the playback call will be taken in order to create a buffer time. The buffer time will be the habituation time between each trial (Figure 1). The nuclear species, nuthatches and chickadees, will lead the mob when attacking the predator. It is predicted that the mixed bird species will return to normal activity after three to five minutes because the nuclear birds tend to drive out the predator in 5 minutes, therefore the nuclear bird will halt the urgency calls, returning the birds to normal activity (Nolan and Lucas, 2009).

       It is predicted that more than 10 birds of two or more species will mob the screech owl playback call because of the birds aggressive tendencies when encountered with a predator and the mixed bird flock is stronger than an individual bird. (Nolan and Lucas, 2009). Based on the results from Figure 2, it is predicted that as the owl screech is played, there will be a 50% increase in birds near the speaker compared to before the playback is played because the mixed bird flock is stronger than an individual bird (Nolan and Lucas, 2009).

       Data of the playback from the Eastern Screech-Owl includes looking at the number of birds from the distances between 0-2, 2-3, 3-5, 5-6, and >6 meters. From 0-2 meters the average number of birds near the speaker was 6.5 birds (Figure 2). From 2-3 meters the average number of birds near the speaker was 6.5 birds (Figure 2). From 3-5 meters away from the speaker the average number of birds was 7.5 (Figure 2). From 5-6 meters away from the speaker the average number of birds was 12.25 (Figure 2). From greater than 6 meters away from the speaker the average number of birds was 24.25 (Figure 2). A general trend that was observed from this figure was that as the distance increases, the number of birds increase.

       It is predicted that the birds will respond to the urgency call given off by the nuclear bird and that they will behave similarly to the way that the birds that responded to the screech owl call. Based on the results from Figure 3, it is predicted these responses will include; the birds calling back to the nuclear bird, the birds pecking aggressively, and swarming looking for a predator because the mixed birds species are territorial and act aggressively when threatened (Lucas and Nolan, 2009).

       It is predicted that fraternity members will show aggressive characteristics towards a non-member male trying to enter their territory because of stereotypical aggressive behaviors when presented with a threat (Calsyn, 1976). Based on the results from Figure 4 we propose that the fraternity members will collectively mob the non member male because they are conforming to their peer group by asserting dominance (Kalof and Cargill, 1991).

       Data from the social observation includes the number of fraternity members approaching the non member and the times in which the members approached. There were 5 trials conducted and each of the trials were averages between 3 group members observing the non member. The first trial was a control and there was one fraternity member that approached the non member at 2 minutes (Figure 3). In the second trial there were 3 fraternity members approaching the non member at 4 minutes (Figure 3). In the third trial there were 3 members approaching the non member at 2 minutes (Figure 3). In the fourth trial there were 4 fraternity members approaching the non member at 3 minutes (Figure 3). Lastly, in the fifth trial, there were 3 fraternity members approaching the non member at 3 minutes (Figure 3).

       We predict that the gene coding for the serotonin receptors in birds and humans will be similar because we expect to see identical base pairs appearing in both gene sequences (Saltzman et al., 1991). If both sequences strongly express similar amino acids, it will demonstrate that the genomes of humans and birds at one point were related and only recently diverged (Saltzman et al., 1991).

Written by: Madison Wilson

Revised by: Madison Wilson

Figure 1. Establishing a buffer time between preliminary playback call trails. The organismal experiment consisted of an Eastern Screech owl playback which is a predator to Titmice, Nuthatch and Chickadee. The playback call was designed to observe the aggressive behaviors in mixed bird species. Each preliminary trial broadcasted the call for a duration of 14 minutes (Nolan and Lucas, 2009). Mobbing which is categorized by aggressive group displays consisting of peaking, looking and erect feathers is a technique used to collectively attack a predator (Arnold, 2000; Dennis et al, 2013). The y-axis is the number of birds involved in mobbing behavior and is plotted against the x-axis which is time that has passed after the preliminary playback call has ended. Recording of the buffer time will be achieved once the birds stop attacking the speaker, time will then stop being recorded. The time for each preliminary trial will be used to calculate an average buffer time. The average time frame from when the playback call ends to when the birds stop mobbing will be used to separate the individual playback trials from one another.

Figure 2. Observed number of mixed birds present at landmarked distance intervals during Eastern Screech owl call. On the Sugar Maple Trail in Fenner Nature Center just passed the Fire Bell a Bluetooth speaker was set up in an exposed log (Rasmussen, 2016). A one-meter piece of twine was used to measure the distances from the log to several significant landmarked trees (Nolan and Lucas, 2009). The marked trees serve as a boundary for the recorded distance intervals. The distances recorded consist of a 0-2m, 2-3m, 3-5m, 5-6m and >6m. After setup, the speaker broadcasted a 14-minute Eastern Screech Owl Call obtained from xeno-canto.org, Eastern Screech Owl (Megascops asio).mp3 (Nolan and Lucas, 2009; Rasmussen, 2016). While the call was played the number of birds in each distance interval in proximity to the speaker was recorded. A total of 4 trials were conducted over a month time frame with a 7-day habituation period between each trial (Nolan and Lucas, 2009). The trials were conducted at 9 am every Saturday morning since it is the period with greatest bird activity (Rasmussen, 2016).

Figure 3. Mixed bird flocks' response time to a nuclear bird urgency call. The Sugar Maple Trail just passed the Fire Bell in Fenner Nature Center will be the destination for this observational study (Rasmussen, 2016). The same exposed log that was used for the previous playback trials will be where the Bluetooth speaker presides. Observers will stand concealed behind shrubbery, careful not to disrupt the investigation but still within view of the speaker and surroundings. A 7-minute Chickadee urgency from xeno-caton.org will be cycled twice to fulfill the 14-minute standardized time from the previous Eastern Screech Owl playback study (Nolan and Lucas, 2009; Rasmussen, 2016). A Chickadee call is broadcasted from the speaker since it is known to be the Nuclear species when presented with predator call (Nolan and Lucas, 2009). A nuclear species is a bird that will facilitate urgency or alarm calls to the surrounding satellite bird species to exhibit aggressive group displays (Nolan and Lucas, 2009). Four trials are to be conducted over a month time frame with a 7-day habituation period after each trial (Nolan and Lucas, 2016). Each trial will commence at 9 am every Saturday morning throughout one month (Rasmussen, 2016).

Figure 4. Mobbing patterns in territorial fraternity members when threatened by a non-member predator. One fraternity house out of the thirty- three total social fraternities on the Michigan State University Campus was chosen at random by an online randomization generator, randomizer.org. A male member of the Lennondary Observers team served as the predator. A predator is considered a male who is not an affiliate of the Greek-life community (Calsyn, 1976). The predator carried an Apple iPhone with a recording app in his pocket and began recording once he entered the fraternity's property. The predator ended the recording once he was no longer on their property. Three observers concealed themselves from the view of the fraternity members but were still within visual range to see the predator. While the predator entered the property the other observers recorded how many of fraternity members approached the non-member till he was removed from the property. After each trial the number of members approaching the predator was averaged. This study was carried out over the course of one month with 7-day habituation period between each trial (Nolan and Lucas, 2009). A total of 4 experimental trials were done and trial 1 was the control for a total of 5 trials. The control consisted of the observers watching one fraternity member enter the property and the number of members that approached him were recorded. The number of members that approached another member were averaged. The member was identified as an actual member by the matching Greek letters of the fraternity worn on his attire.

Figure 5.Spartan Spirit Biology Student News Interviews the Lennondary Observers