To act as a control and determine if vocal communication played a more important role than body communication, a playback experiment involving Michigan State University students was conducted in Holmes Hall and Snyder-Phillips Dining Halls at Michigan State University. The experiment was conducted during a 45-minute time span once a week for nine weeks. The date, time, cafeteria, and all qualitative observations were recorded. A speaker was not used because students would not be affected by an outside voice, so people acted as the speaker. Prior to arriving at the cafeteria, volunteers were contacted in order to set up a time to observe their group during a normal meal time. The volunteer was instructed to not disclose any information about the experiment to any outside parties. They were instructed to use the Voice Memo application on the IPhone to document the vocalizations during the meal. After arriving at the cafeteria, the number of subjects sitting at the observed table was recorded. Observers were sitting at tables near the subjects, while also eating to maintain consistency with their environment, and recording the subjects with the photograph/video iPhone application with a concealed phone. Observations were made regarding any physical communication (i.e. head turning) that accompanied the leaving vocalization. To act as a positive control, the volunteer acted as a speaker, and after thirty minutes, vocally signaled a departing phrase. This acted as a positive control because this phrase is likely to induce others to leave the cafeteria with the volunteer. During the thirty minutes, the volunteer acted as a negative control, by engaging in normal conversation that would not induce others to leave the cafeteria. After the observations were recorded, the standard deviation was used to find the percent of subjects present that left the cafeteria after the leaving vocalization playback occurred, and to determine if the amount of body communication decreased when sounds were heard.

Gene Analysis

           Gene analysis needed to be performed to determine that the Dopamine Beta Hydroxylase Gene was present in Mallard Ducks and Humans, proving that there is a connection between Mallard Ducks and Humans. This began with DNA Purification, followed by PCR, and gel electrophoresis.

DNA Purification

           When the mallard duck blood was centrifuged, the temperature was measured between 15-25 degrees Celsius. 20 microliters of proteinase K was pipetted into a microtube, the position of the blood was identified with a well plate register. 200 microliters of a phosphate buffer solution suspended the blood. 20 microliters of proteinase K was added again to the samples as well as 200 microliters of Buffer AL. Each tube was sealed with a cap. A cover was placed over the microtubes and for 15 seconds the racks of tubes were shaken. The caps were then centrifuged until it reached 3000 rpm. Incubation occurred at 56 degrees Celsius for 10 minutes, and during this time, a weight remained on top of the caps, mixing from time to time. After the caps were taken off, 200 microliters of ethanol was added to the current sample. Once again the microtubes were sealed off with caps with a clear cover on top and shaken for 15 seconds (Qiagen Inc., 2006). The sample was centrifuged until it reached 3000 rpm. Two DNeasy 96 plates given in the qiagen kit were placed on top of the S-Blocks and marked. The lysis mixture was dispensed into the wells of the DNeasy plates and were sealed with an AirPore Tape Sheet. The sample was centrifuged for 4 minutes at 6000 rpm. The tape was removed, and 500 microliters of Buffer AW1 was added. Another tape sheet was added to the plate and the sample was centrifuged at 6000 rpm for 2 minutes. The tape was again removed, and 500 microliters of Buffer AW2 solution was added. The solution was centrifuged at 6000 rpm for 15 minutes. Plates were placed on an Elution Microtubes RS. 200 microliters of Buffer AE were added for elution to occur. Incubation at room temperature occurred for one minute, using AirPore Tape Sheets to seal the samples. The sample was then centrifuged at 6000 rpm for 4 minutes. These methods were repeated for the human blood. (Qiagen Inc., 2006).

Primers

           For annealing to take place in PCR, the correct primers were chosen to bind a single strand of DNA from the blood of the mallard duck taken from a laboratory on the campus of Michigan State University and hair from a human. Twenty base pairs were identified in the human and mallard duck genomes to create the primers. Integrated DNA Technologies (IDT) was the company that produced these primers. Annealing temperature was calculated with the formula provided in the lab manual, which is: Tm=64.9o C + 41oC x (number of Gs and Cs in the primer-16.4)/N, where N is the length of the primer (Igert, et al., 2015).

PCR

           The Type-It Microsatellite PCR kit was used to obtain the separate animals genotype. Equal concentrations of all primers were used. The 2x Type-It Multiplex PCR Master Mix, template DNA, RNase-free water, and primer mix were all thawed and placed into PCR tubes along with the template DNA. PCR tubes were put into the thermocycler. Initial activation occurred for five minutes at 95 degrees Celsius, followed by 28 cycles of denaturation at 95 degrees Celsius for 30 seconds, annealing at 60 degrees Celsius for 90 seconds, and extension at 72 degrees Celsius for 30 seconds. This was followed by a final extension for 30 minutes at 60 degrees Celsius. The sample was then analyzed using the capillary sequencer instrument (Qiagen Inc., 2009).

Agarose Gel Electrophoresis

           0.4 grams of agarose was combined with 40 mL of 1X TBE Solution and brought to a boil with a microwave. The solution was cooled to a comfortable touch. 4 microliters of SybrSafe was added to the solution and swirled until the color changed. The mixture was poured into a casting tray, and a comb was placed to form the wells of the gel. After the gel solidified, the DNA samples of the duck and humans were loaded into the wells, as well as a Kb ladder to act as a control and indicator of molecular weight, where the wells are closest to the negative electrode. 1X TBE Solution was added to the device to flood the gel. The black and red electrodes were connected to the negative and positive terminals, respectively. A switch was flipped to turn on a power source and voltage was set to 100 volts. The gels were run until the loading dye was three-quarters down the gel. The power source was turned off, and the gel was placed under a UV light to observe the results. The ladder was used to find the relative molecular weight of the bands shown, and the band sizes were recorded.

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Results

Responsibility by: Sabrina Cleis

Mallard Ducks

           As pigeons approach flight, there is an increased neck stretching or looking upwards (Davis, 1975), which is also true for head-shaking in swans (Black, 1988). This shows there is a commonality among different types of birds for preflight communication. We predict that head tossing frequency in Mallard Ducks will increase approaching flight, and especially in a group of four or more ducks (Figure 1) because the total number of head-tosses before flight for a single goose was found to be 4.5 and was found to be 171 in families of four (Raveling, 1969). Head tossing was found to reach its peak intensity right before flight in geese, leading us to believe the same results will be true for Mallard Ducks (Raveling, 1969).

Humans

           Humans replicate interactive behaviors in order to communicate with one another, which is seen in studies done to improve communication (Mohammad & Nishida, 2015). When a communication behavior is displayed, there are steps taken to learn the meaning and to replicate it in a homologous situation. We predict that head tossing frequency in humans will increase approaching departure from the cafeteria, and especially in a group of four or more subjects (Figure 2) because humans learn and mimic social behaviors for communication (Mohammad & Nishida, 2015), and research has shown an increase in amount of movement before departure from an area (Martino-Saltzman et al., 1991).

Dopamine Beta-Hydroxylase Gene

           We predict that the dopamine beta-hydroxylase gene (DBH) found in human and Mallard Duck DNA will have the same molecular weight (Figure 3) because data shows that DBH was found in both species and they both have a molecular mass of 1869.755 base pairs (DBH Gene, 2016). The molecular weights will align at the same point on the DNA ladder of the human and the duck (Bio-Rad, 2016).

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References

Responsibility by: Maggie McDonald

Addgene. Agarose Gel Electrophoresis. https://www.addgene.org/plasmid-protocols/gel-electrophoresis/, last accessed 10/18/16.

Anonymous. DBH Gene. 2016. Retrieved November 20, 2016, from http://www.genecards.org/cgi-bin/carddisp.pl?gene=DBH-AS1

Anonymous. QIAGEN. 2006. DNeasy Blood & Tissue Handbook. https://www.qiagen.com/us/resources/resourcedetail?id=6b09dfb8-6319-464d-996c-79e8c7045a50&lang=en, last accessed 10/19/16.

Anonymous. QIAGEN. 2011. DNeasy Blood & Tissue Kit. https://www.qiagen.com/us/shop/sample-technologies/dna/dneasy-blood-and-tissue-kit/#orderinginformation, last accessed 10/18/16.

Anonymous. QIAGEN. 2009. Type-it Microsatellite PCR Handbook. https://www.qiagen.com/us/resources/resourcedetail?id=d6135896-0466-4d9d-aeef-1c4f23f8964e&lang=en, last accessed 10/19/16.

Black, J.M. 1988. Preflight Signalling in Swans: A Mechanism for Group Cohesion and Flock Formation. Ethology. 79: 143-157.

Davis, J.M. 1975. Socially Induced Flight Reactions in Pigeons. Animal Behaviour. 23: 597-601.

Dunbar, R. I. 1993. Coevolution of neocortical size, group size and language in humans. Behavioral and brain sciences. 16(04): 681-694.

Garland, E. M., D. Robertson. 2015. Dopamine Beta-Hydroxylase Deficiency. Genereviews. 1-62.

Gel Electrophoresis- Teacher Instructions. n.d. http://www.colorado.edu/Outreach/BSI/pdfs/electrophoresis_teacher.pdf, last accessed 10/19/16.

Howard, L. 2003. "Anatidae" (On-line), Animal Diversity Web. Accessed November 19, 2016 at http://animaldiversity.org/accounts/Anatidae/

Igert, et al. 2015. LB144 Course Pack. MSU Printing Services, East Fee Hall. Michigan State University, East Lansing, MI.

Jones, O.E., Williams, C.K., and Castelli, P.M. 2014. A 24-Hour Time-Energy Budget for Wintering American Black Ducks (Anas rubripes) and its Comparison to Allometric Estimations. Waterbirds. 37(3): 264-273.

Martino-Saltzman, D., B.B. Blash, R.D. Morris, L.W. McNeal. 1991. Travel behavior of nursing home residents perceived as wanderers and nonwanderers. The Gerontologist. 31(5): 666-672.

Mohammad, Y. & Nishida, T. 2015. Learning interaction protocols by mimicking understanding and reproducing human interactive behavior. Pattern Recognition Letters. 66: 62-70.

Mowbray, T. B., C. R. Ely, J. S. Sedinger, and R. E. Trost. 2002. The Birds of North America Web Site. https://birdsna.org/Species-Account/bna/species/cangoo, last accessed 9/21/16

Nussey S, Whitehead S. Endocrinology: An Integrated Approach. Oxford: BIOS Scientific Publishers; 2001. Chapter 4, The adrenal gland. Available from: https://www.ncbi.nlm.nih.gov/books/NBK26/

Raveling, D.G. 1969. Preflight and Flight Behavior of Canada Geese. Auk. 86: 671-681.

Rosenzweig, M. R., Leiman, A. L., & Breedlove, S. M. (1996). Biological psychology. Sinauer Associates.

Senard, J. M., & Rouet, P. 2006. Dopamine beta-hydroxylase deficiency. Orphanet journal of rare diseases, 1(1), 1.

Sigma-Aldrich. 2016. Standard PCR protocol. http://www.sigmaaldrich.com/technical-documents/protocols/biology/standard-pcr.html, last accessed 10/18/16.

Tingen, C. 2016. What is PCR? http://nubio.northwestern.edu/video/what-pcr, last accessed 10/19/16.

The Cornell Lab of Ornithology. 2015. Mallard. https://www.allaboutbirds.org/guide/Mallard/ lifehistory, last accessed 10/18/16.

Xu, Q. et al. 2016. The dopamine β-hydroxylase gene in Chinese goose (Anas cygnoides): cloning, characterization, and expression during the reproductive cycle. BMC Genetics. 17(48): 1-9.

Zhang, Z., Schwartz, S., Wagner, L., and Miller, W. 2000. "A greedy algorithm for aligning DNA sequences", J Comput Biol 2000; 7(1-2):203-14.

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Predicted Figures

Responsibility by: Sabrina Cleis

A. B.
C.
Figure 1. Predicted results of Mallard Duck observations and playback experiment. A. GIF showing site of observation and playback experiment for Mallard Ducks. Observations occurred for one hour, twice a week for nine weeks by the Red Cedar River near the Michigan State University Main Library. B. The number of ducks present and number of head tosses (rapid movement of the head from left to right) were recorded while observing Mallard Ducks near the Michigan State University Main Library on the Red Cedar River. The time at which each action occurred was recorded to determine the time and frequency at which the communication behavior took place before flight. After the observational period concluded, the average head tosses per duck in relation to time approaching flight were compared between groups. We predict that head tossing frequency in Mallard Ducks will increase approaching flight, and especially in a group of four or more ducks because the total number of head-tosses before flight for a single goose was found to be 4.5 and was found to be 171 in families of four (Raveling, 1969). Future work would include performing a correlation test to determine the impact of number of ducks present on social communication approaching flight. C. A playback experiment was conducted in the same location as the observational study of Mallard Ducks. The number of ducks present in a 20 meter range was recorded prior to the initiation of the synthetic vocalization. Once the vocalization finished playing for 30 seconds from a Bluetooth speaker, the response vocalizations were recorded using a Microphone iPhone attachment. The number of ducks departing the 20 meter range as a result of the synthetic vocalizations was recorded and also captured on video using a smartphone. This procedure was repeated with the use of human clapping as a negative control. After the observations were recorded, the percent of ducks that left the parameter after the playback vocalization was found. We predict that a synthetic duck vocalization will elicit departure in Mallard Ducks because vocalizations have been shown to communicate leaving behaviors in Canadian Geese (Raveling, 1969). Future work would include performing a chi square test for independence to determine the impact of playback vocalization on leaving behavior in different groups of ducks.

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A. B.

C.
Figure 2. Predicted and observed results from cafeteria observations of humans. A. GIF of Holmes Hall Cafeteria. Observations occurred for one hour, twice a week for nine weeks in Holmes Hall and Snyder Phillips Hall cafeterias at Michigan State University. B. The number of head movements (a deep turn of the head to the left or right) was recorded in various group settings. Observations began when the first head movement was observed, and ended when the group departed from the cafeteria. Our results indicate that in groups with a greater number of people, there is an increased amount of head movements. We predict that there will be an increase in the number of head movements in groups with a greater amount of individuals because studies show that humans learn and mimic social behaviors for communication (Mohammad & Nishida, 2015), and research has shown an increase in amount of movement before departure from an area (Martino-Saltzman et al., 1991). Future work would include performing a correlation test to determine the impact of number of people present on social communication approaching departure. C. Playback experiments were performed to determine if a departing vocalization affected the amount of communication behaviors made by humans before departure from the cafeteria. A volunteer acted as a positive control; they sat down to eat and then waited thirty minutes before saying a departing phrase, such as bye or gotta go. Meanwhile, not saying any departing phrases acted as a negative control. We predict that when the positive control is enacted, it will elicit departure from the cafeteria by the humans present in the group. This is because Joseph Dunbar concluded that because the neocortex in humans is larger, humans are more comfortable in greater groups of people, therefore students want to depart the cafeteria together, to remain in larger groups (Dunbar, 1993). This would mean that humans are affected by departing phrases, and affect the amount of physical communication made.

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Figure 3. Predicted results of agarose gel electrophoresis after PCR amplifies the copies of DNA segments. A primer was found from human DNA for dopamine beta hydroxylase in exon 3. The forward primer 5 prime-GCTGCAGGACGGC-3 prime. The reverse primer was 3 prime-GAAGCCCTTTGGAAGCTCCT-5 prime (Zhang et al., 2000). The following primers were used during the PCR. Temperatures were set for denaturation, annealing, extension, and final extension for the duck and human PCR runs. Denaturation occurred for 30 seconds at 95 degrees Celsius. Annealing temperature was calculated from the following equation: Tm= 64.9°C + 41°C * (number of G and Cs in primer -16.4)/N (Igert, et al., 2015). Calculated possible temperatures were 48.5 degrees Celsius for the human forward primer and 53.83 degrees Celsius for the reverse primer. An annealing temperature of 47 degrees Celsius was used. In ducks the forward primer was 5 prime-CGGGCACAGCTCGTATTTTG-3 prime and the reverse primer was 3 prime-TCTCCTGGCTGGGGATAACA-5 prime. Melting temperature was calculated for the forward and reverse primers at 55.8 and 62.03 degrees Celsius, respectively. The annealing temperature was set to 55 degrees Celsius. Extension occurred in both for 30 seconds at 72 degrees Celsius. Final extension occurred for 30 minutes at 60 degrees Celsius, totaling 28 cycles. Gel electrophoresis was run and the gel was placed under UV light to observe the results (Qiagen Inc., 2009). The molecular weights on the side ladder align with the duck and human bands at 1864 bp.

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Figure 4. Documentary showcasing LB 144 research project. Video recordings were taken from early September until mid November documenting the research process undergone during the 2016 Fall Semester of LB 144. The footage was then made into a film showcasing the process.