Video Recording of Human and Mallard Duck Response Rate Which Increase with Higher Environmental Sound Frequencies
By: Davin Hami, Kate Hammond, Emily Liddicoat, and Rachel Best
LB 144 Organismal Biology
Wednesday 11:30 AM
Morgan Kiryakoza and George Hyde
11/22/2016
https://www.youtube.com/watch?v=4XDlWbletIw&t
www.msu.edu/~hamidavi/
Introduction
Responsibility by: Kate Hammond
Both male and female mallard ducks (Anas platyrhynchos) with hearing ability respond to acoustic stimuli, just as humans (Homo sapiens) do. Mallard ducks often live and travel in groups in which they communicate; when certain audible environmental stimuli are presented, the ducks can communicate and react in various ways due to their danger complexes (Goudie et al., 2004). A stimulus is something that can elicit a physiological response in an organism, triggering sensitive sensory receptors (Hine et al., 2015). Acoustic environmental stimuli can range from the pattering of rain, to the roar of thunder, to humans speaking in the environment: all of these can draw attention and reactions from organisms who hear them. Frequency (measured in hertz) is scientifically defined as number of cycles of a wave moving past an observation point per second, and is commonly correlated with the term "pitch" (Cleveland et al., 2014). Humans speaking, rain pattering, and thunder roaring all increase in frequency respectively, and since noise is ubiquitous, animals and humans are always disturbed by environmental noises such as these (Brumm et al., 2005).
All species of mallard ducks have their own ways of communicating behaviors within their respective social groups. Human and duck ears are able to detect several different sound frequencies, and the hearing of both can be quite sensitive (McAlpine et al., 2001). Hearing loss in humans can result from the natural and chronic process of aging, from the continuous exposure to specifically-pitched noises, and from having hereditarily sensitive ears (McCabe, 1979). If a duck does not display the correct behaviors upon being exposed to an auditory environmental stimulus, it is likely that the duck has had a loss of hearing or has developed a sensitive response to certain frequencies (Crowell et al., 2016). This is especially important in the environment, because environmental dangers such as a thunderous roar signal a coming storm, which indicates a need to prepare and find shelter for in severe cases (Sheboygan, 2007). Ducks act in the same fashion; the ducks who do not display certain behaviors in response to certain sounds typically do not fit into their social groups either, as many ducks rely on each other's hearing abilities and auditory cues to detect danger (Dessborn et al., 2012).
Upon initial observation, mallard ducks and humans display certain behaviors when they hear rain, thunder and human voices (Dahlgren, 1992). The ducks respond in a sensitive manner to the stimuli in the environment, and different basal levels of function are established and maintained according to the prevailing level of environmental stimulation (Welch et al., 1969), and humans respond with similar behaviors (Dahlgren, 1992). There are many behaviors which ducks display in response to noise stimulation (Goudie et al., 2004). The observed behaviors discussed include agonism, such as aggression; courtship; feeding; resting; peering, which includes gazing into the water (with ducks) or gazing into the distance (with humans); locomotion behaviors include any movement; preening includes cleaning or shaking feathers (with ducks) or fixing up appearance and clothes (with humans); social; vigilant, which includes keeping a lookout for a partner; and alert behaviors have stretched heads and tense bodies (Goudie et al., 2004). Goudie found that ducks increased their alert behavior during jet fly-overs at a statistically significant level. It was also found that jet flyovers were much higher in frequency than the normal background noise, so the onset of alert behaviors was attributed to the high frequencies of the jet flyovers (Goudie et al., 2004). Using rain, thunder, and humans speaking as a playback experiment in place of military jets to observe mallard duck behaviors on the Red Cedar River should elicit similar results. Rain has had a positive effect on duck population; it was shown that duck population increases when it rained more frequently (Richardson, 2008). Thunder can indicate danger to ducks and can increase the response behavior of agitation and locomotion (Sheboygan, 2007). Human activities along the river can force ducks to move away from feeding grounds, increase a duck's motivation to fly away, and can lower how productive the duck is since it is now being provided food (Dahlgren, 1992).
SLC26A5 (solute carrier family 26 member 5) is a gene that encodes the protein prestin, a transmembrane protein of the cochlear outer ear hair cell; prestin is required for cochlear hair cell activity, which is how sound is heard (Minor et al., 2009). SLC26A5 is composed of 2,343 base pairs (bp) and is located on chromosome 7 (Nikiforov et al., 2012). SLC26A5 is a voltage-dependent motor protein, which plays an important role in how organisms are selective with the frequencies that they hear and how sensitive their hearing is (Homma et al., 2010). The gene also helps depict frequencies of the noises happening in the environment. Without this gene, major hearing impairment or even deafness can result. Mallard ducks more readily respond to high frequency acoustic cues; the higher the frequency of the surroundings, the more aggressive and alert mallard ducks become (Therrien, 2015). Organisms respond to noises by exhibiting alert behaviors, and these behaviors increase as the frequency of the noise increase (Goudie et al., 2004). Rain, thunder, and humans speaking recordings are being used in this study to determine the frequency in which mallards and humans display the most about of alert behaviors, as well as the other nine aforementioned behaviors. The recorded, natural frequency is to be used in addition to an altered frequency of 300 Hz for all three sounds. Higher frequencies will cause mallard ducks and humans to display more alert behaviors due to the SLC16A5 gene. We predict that as the frequency of sound increases the mallard ducks will become more alert to their surroundings because they will demonstrate behaviors such as tension, agitation, and head stretching upward at two times the average rate, as the frequency doubles (Goudie et al., 2004). We also predict that if there is no sound coming from the speakers the ducks will have less aggressive behaviors than if the speakers are playing rain, thunder, or human sounds because the ducks will not be stimulated or disturbed from the current things they are doing if there is no sound occurring (Goudie et al., 2004). For the genetic testing, we predict that humans and ducks will share the SLC26A5 gene because SLC26A5 is extremely important in frequency sensitivity in humans (Homma et al., 2010), and likely impacts the frequency sensitivity of ducks as well.
Methods
Responsibility by: Rachel Best
Duck Response
To conduct the playback experiment of mallard ducks, the materials used were an iPhone 6 camera and a Canon PowerShot SX410 camera. They were used to record videos and capture photos of the ducks and humans. While the playbacks were being performed, a notebook was used for a place to write down any observations. The videos and photos were edited using Final Cut Pro and used in an educational documentary. To conduct the playback experiment, two bluetooth Bose Soundlink speakers were used. The bluetooth speakers were randomly placed one meter from the Red Cedar River's edge in order to make sure the ducks did not become custom to hearing the sound in one certain location. They were placed on the north and south side of the river and were constantly moved after each trial so that the ducks did not become accustomed to the sound coming from the same location. In order to make sure the ducks did not become attracted to a certain speaker, one speaker at a time was randomly chosen to play the sound. As a negative control, the ducks were observed when no sound was playing from the speakers. The negative control was used to compare the different behaviors displayed when no stimuli was presented, in comparison to the behaviors displayed as the frequency of each stimuli increased. The different audio sounds used for the playbacks were first recorded in nature: the sounds of rain, thunder, and humans speaking were recorded during a thunderstorm using the iPhone app "Voice Memos" that comes on all iPhones. The frequency changer software, Audacity, was then used to change the frequency of all three playback sounds to 300 Hertz. The frequency was converted to the same level to understand if frequency has an impact on the sensitivity of hearing, shown through increased alert and aggressive behaviors, or if the specific noise leads to the certain behaviors. The frequency audio was then exported as an MP3 file and used in the playback methods stated below.
Research was conducted during the fall 2016 semester at Michigan State University in East Lansing, Michigan. The mallard ducks were observed through a series of playback experiments, at the Red Cedar River rapids (42 degrees 43' 43.7556'' N, 84 degrees 28' 54.9624'' W) located between Wells Hall and the MSU Main Library. The data was quantified twice a week, for two hours each day. The days and times each week were randomly chosen to rule out any human bias. The mallard ducks were monitored in fifteen second time periods for one behavior, until all ten behaviors were observed while the playback calls at the varying frequencies were played (humans speaking, thunder, and rain) to make sure every behavior was observed and accounted for. Three out of the four members in the group observed the ducks or humans, while the final member controlled the stopwatch (standard iPhone stopwatch application) and recorded the observations with an iPhone 6 video camera and/or photographs with the Canon PowerShot SX410. The frequency of the noise from the randomized speaker was placed one meter from the Red Cedar River's edge. The ten categories of behaviors were separately observed and recorded in a laboratory notebook. The ten behaviors observed were agonism, alert, courtship, feeding, peering, locomotion, preening, rest, social, and vigilant. The behaviors were recorded in a table, which accounted for the total ducks present and the number of ducks presenting each type of behavior for every fifteen second interval.
Human Response
The human experiment was performed the same way as the duck experiment, using the same materials, except humans were observed at the CATA bus station instead of the Red Cedar River. The CATA station was used for observations, because just like the ducks congregate at the rapids, humans congregate at the CATA station. The two speakers were placed on the ground at the CATA bus station. The speakers were put in places that were not easily visible, so the subjects did not know where the sound was coming from and pure reactions could be observed. Observations began on September 18th and ended on November 18th, and they took place for one hour twice a week, every Monday at 10 am and every Friday at 12 pm. The behaviors were recorded for the duration of the observation, categorized by the ten types of behaviors. Each behavior was observed separately for fifteen seconds, with the speaker playing no sound to act as a negative control until all behaviors were observed. This is a negative control because alert, aggressive, and vigilant behaviors are not expected to be seen at a high rate when there is no sound coming from the speaker. During each playback trial, the environmental stimuli (rain, thunder, and humans speaking) were played at their natural frequency, as recorded in nature, and each behavior was observed for fifteen seconds. The frequencies were all converted to 300 Hz to see if it the frequency or the specific noise stimulus has an impact on sensitivity of hearing shown by alert behaviors. Multiple trials were done using the same methods for the control and playback experiments.
Calculations and Data Analysis
In order to account for the changing number of ducks and humans observed at the Red Cedar River and the CATA bus station each day and during each trial, the average percent of subjects performing a certain behavior during a playback at a certain level of frequency was quantitated. The average number of ducks or humans performing a certain behavior was calculated for each fifteen second time period by taking the total number of subjects which performed that behavior, and dividing by the total number of subjects present during the observation. This number was then multiplied by 100 to get a percentage. Once the percentage of subjects performing the behavior for each 15 second time increment was calculated, each trial's percentages were added up and then divided by how many trials were performed. This gave the total percent of subjects performing a behavior at one frequency. The same calculations were used for every playback and control test. To analyze the data for both the duck and human observations, a chi-squared test of independence was performed. The test of independence was performed because it was deemed most relevant when comparing two populations through categorical variables. The degree of freedom was found using the chi-squared distribution table in order to find the p-value (Kubiak et al., 2009).
Genome Extraction
In the genetic portion of this study, mallard duck DNA was obtained from Culver Duck Farm (Middlebury, IN), as well as human DNA which was collected from the the Erik Shapiro lab at Michigan State University (East Lansing, MI). These were used to test for the presence of the SLC26A5 gene in the duck and human genome. A thermocycler and PrimePCR SYBR Green Assay: SLC26A5 from Bio-Rad (Hercules, CA) was also used to perform Polymerase Chain Reaction (PCR) amplification on the extracted DNA. A standard gel electrophoresis set up (electrodes, voltage source, casting tray, gel box, and well combs) was used to perform gel electrophoresis on the PCR amplification products.
The mallard and human DNA were prepared and analyzed by PCR within 5 hours of collection. The DNA extract for the PCR-based assays was assessed by amplifying the SLC26A5 gene, which was amplified by PCR using 6 microliters of the primer PrimePCR SYBR Green Assay: SLC26A5. PCR was carried out in 20 microliter total reaction volumes and the reaction mixture was heated in the thermocycler to 94 degrees Celsius for 5 minutes to denature the DNA, followed by 5 minutes at 60 degrees Celsius to allow the primers to anneal to the complementary sequences. The temperature was then set at 72 degrees Celsius to allow the Taq polymerase to attach at each priming set to synthesize the new DNA. This is repeated for 30 cycles to achieve over 1 billion copies of the SLC26A5 gene. The PCR products were then subjected to gel electrophoresis as follows (Ghatak et al., 2013).
To prepare the 1% agarose gel, 0.5 g of agarose obtained from Lonza (Basel, Switzerland) was mixed with 5 mL of 10X Tris-acetate-EDTA (TAE) from ThermoFisher (Waltham, MA), and 45 mL of DH2O. The mixture microwaved for 2 minutes, ensuring that no over-boiling occurred. The agarose solution was allowed to cool to 60 degrees C, 5 microliters of ethidium bromide dye from ThermoFisher (Waltham, MA) was added (1 microliter of dye for every 10 mL of gel), then the solution was poured into the gel box with the well comb in place at the negative end. The well comb ensured proper wells were formed in the gel as it solidified. The gel set for 30 minutes at room temperature to ensure complete solidification. Once solidified, the comb was carefully removed from the gel (Smith et al., 1996).
To perform the gel electrophoresis, the gel box was filled with just enough 1X TAE to cover the wells. All 20 microliters of the PCR products were placed in the wells as well as 20 microliters of the ladder: well 1 contained Precision Plus Protein Dual Color Standards molecular weight ladder from Bio-Rad (Hercules, CA), well 2 contained the duck sample, and well 3 contained the human sample. There was no need to stain as the 2X PCR Master Mix II contained dye that allowed direct loading of PCR products onto the agarose gel. The lid to the machine was shut and 80 volts were applied for 30 minutes. Once the current ceased and the machine was unplugged, the gel was removed from the gel box and the products were analyzed (Smith et al., 1996).
The results were analyzed by comparing the bands within the gel. The bands represent there is a gene in the DNA amplified by the PCR at a certain size. It is known that the gene SLC26A5 lies within the human genome and contains 2,343 base pairs (bp) (Nikiforov et al., 2012). The bands that correspond with well two and three were measured against the ladder at approximately 2,300 bp, a presence of a band would be analyzed to confirm the presence of the SLC26A5 gene in both mallards and humans.
Results
Responsibility by: Emily Liddicoat
We predict that as the frequency of sound increases, the mallard ducks will become more tense and agitated by demonstrating more alert, aggressive, and vigilant behaviors, because the frequency of sound increases the sensitivity of hearing (Goudie et al., 2004). Based on Goudie's experiment, when the frequency was increased due to jet engines flying over the ducks habitat, the ducks demonstrated more alert, aggressive, and vigilant behaviors such as startle and panicked responses (Goudie et al., 2004). We also expect to see the same reaction as the frequency of environmental stimuli are increased (Figure 1A). Alert behavior is predicted to rise as the noise stimulus frequency increases; therefore, humans speaking should have the highest percentage of alert, aggressive and vigilant behaviors because it has the highest frequency in nature (Figure 1A). We predict that if the frequency of each environmental stimuli are converted to the same frequency, then each noise stimulus will yield the same behavioral results because the behavioral response is dependent on the frequency level, measured in hertz (Goudie et al., 2004). We expect that changing the environmental sounds of rain pattering, humans speaking, and thunder roaring to the 300 Hz will lead to similar behavioral responses for all three stimuli, showing that frequency must lead to a greater sensitivity of hearing (Figure 1B).
We predict that humans will respond with more alert behaviors when exposed to environmental stimuli with higher sound frequencies, because published results indicate that when humans are exposed to various environmental stimuli, they are disturbed by the noises and their frequencies (Brumm et al., 2005). When Goudie performed his experiment on mallard ducks, he found that the increased frequency of jet engines flying over the ducks resulted in the ducks being more alert to danger and in an increase of behavioral responses (Goudie et al., 2004). We expect to find the same results in humans, because the behaviors exhibited by ducks can also be exhibited by humans (Brumm et al., 2005). The behaviors that are expected to increase when exposed to stimuli with high frequencies, such as humans speaking, are agonism, alert, and vigilance (Figure 2A). The expected behaviors to be displayed when humans are exposed to stimuli with lower frequencies, such as rain pattering, are social and locomotion (Figure 2B). We predict that if rain pattering, thunder, and humans speaking are all converted to the same frequency, then similar behavioral responses will be observed because frequency of sound leads to hearing sensitivity (Brumm et al., 2005). When the frequency is converted to 300 Hz for all three stimuli, we expect to see high and equal rates of alert, aggressive, and vigilant behaviors for rain pattering, thunder, and humans speaking, showing that frequency affects these behaviors, not just the noise stimulus (Figure 2B).
We predict that if there is no sound coming from the speakers, then the ducks and humans will have less aggressive behaviors than if the speakers are playing rain, thunder, or humans speaking because the ducks and humans will not be stimulated or disturbed if there is no playback occurring (Goudie et al., 2004). The ducks will show minimal aggressive reactions when the speaker has no sound playing because they have no stimulus to trigger an aggressive response (Figure 3A). We predict that when the speakers are put out with no sound coming from them, the feeding and courtship behaviors will be greater than when the speakers play sound because no sound results in low rates of disturbance and a higher rate of natural behaviors (Goudie et al., 2004). Goudie observed an increase in courtship and feeding when jet planes were not flying over the ducks heads; we think that the speaker with no sound will show the same results in humans (Figure 3C).
For the genetic testing, we predict that humans and ducks will share the SLC26A5 gene because humans and ducks respond with similar alert behaviors to high sound frequencies (Goudie et al., 2004). The band in well two (duck sample) and well three (human sample) is expected to line up precisely between 2025 base pairs and 2700 base pairs with well one, which is the molecular ladder (Figure 4). This indicates that the SLC26A5 gene, which is roughly around 2,343 base pairs, is in both species genomes (Figure 4). We predict that the band will fall between 2025 base pairs and 2700 base pairs because the gene's molecular size is 2,343 base pairs (Nikiforov et al., 2012).
References
Brumm, H. and H. Slabbekoorn. 2005. Acoustic Communication in Noise. Advances in the Study of Behavior. 35: 151-209.
Cleveland, C. J. and C. G. Morris. 2015. Dictionary of Energy - 2nd ed. Elsevier Science & Technology Books, Amsterdam.
Crowell, S. E., A. M. Wells-Berlin, R. E. Therrien, S. E. Yannuzzi, and C. E. Carr. 2016. In-air hearing of a diving duck: A comparison of psychoacoustic and auditory brainstem response thresholds. The Journal of the Acoustical Society of America. 139: 3001-3008.
Dahlgren, D. B. 1992. Human Disturbances of Waterfowl: Causes, Effects, and management. U.S. Fish and Wildlife Service. 13: 1-8.
Dessborn, L., G. Englund, J. Elmberg, and C. Arzel. 2012. Innate responses of mallard ducklings towards aerial, aquatic, and terrestrial predator. Behaviour. 149(13-14): 1299-1317.
Gard, C. 2003. Thunderstorms aren't all noise. Current Health. 27(2): 29-31.
Ghatak, S., R. B. Muthukumaran, and S. K. Nachimuthu. 2013. A simple method of genomic DNA extraction from human samples for PCR-RFLP analysis. J Biomol Tech. 24(4): 224-31.
Goudie, R. I. and I. L. Jones. 2004. Dose-response relationships of harlequin duck behaviour to noise from low-level military jet over-flights in central Labrador. Environmental Conservation. 31(4): 289-298.
Hine, R. and E. Martin. 2015. A Dictionary of Biology - 7th ed. Oxford University Press, Oxford.
Holmes, C. R., M. Brook, P. Krehbiel, and R. McCrory. 1971. On the power spectrum and mechanism of thunder. Journal of Geophysical Research. 76: 2106-2115.
Homma, K., K. K. Miller, C. T. Anderson, S. Sengupta, G. Du, S. Aguinaga, M. Cheatham, P. Dallos, and J. Zheng. 2010. Interaction between CFTR and prestin (SLC26A5). Biochimica et Biophysica Acta (BBA)-Biomembranes. 1798(6): 1029-1040.
Kubiak, T. M. and Benbow, D. W. 2009. Certified Six Sigma Black Belt Handbook - 2nd ed. ASQ Quality Press, Milwaukee.
McAlpine, D., D. Jiang, A. R. Palmer. 2001. A neural code for low-frequency sound localization in mammals. Nature Neuroscience. 4: 396-401.
McCabe, B. F. 1979. Autoimmune Sensorineural Hearing Loss. Ann Otol Rhinol Laryngol. 88(5): 585-589.
Minor, J. S. and Y.H. Tang. 2009. DNA Sequence Analysis of SLC26A5, Encoding Prestin, in a Patient-Control Cohort: Identification of Fourteen Novel DNA Sequence Variations. PloS one. 6: 1-8.
NCBI. 2013. Database resources of the National Center for Biotechnology Information. Nucleic Acids Research. 41: D8.
Nikiforov, Y. E., P. W. Biddinger, and L. DR Thompson. 2012. Diagnostic pathology and molecular genetics of the thyroid: a comprehensive guide for practicing thyroid pathology. Lippincott Williams & Wilkins, Philadelphia.
Omland, K. E. 1996. Female Mallard Mating Preferences for Multiple Male Ornaments: I. Natural Variation. Behavioral Ecology and Sociobiology. 39(6): 353-360.
Richardson, C.J. 2008. The National Wetlands Newsletter. Environmental Law Institute. 30(2): 35.
Sanfilippo, P. G., Casson, Yazar, S., D. A. Mackey, and A. W. Hewitt. 2015. Review of null hypothesis significance testing in the ophthalmic literature: are most 'significant' P values false positives? Clinical & Experimental Ophthalmology 44(1): 52-61.
Smith, D. R. 1996. Agarose gel electrophoresis. Basic DNA and RNA Protocols: 17-21
Therrin, R. E. and S. E. Crowell. 2015. In-air hearing of a diving duck: A comparison of psychoacoustic and auditory brainstem response thresholds. Cross Mark. 139: 10-80.
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Predicted Figures
Responsibility by: Davin Hami
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Predicted Figure 1. Predicted results of the average percent of mallard ducks performing one of ten different behaviors in response to the different frequencies, and one constant frequency, of environmental stimuli: rain, thunder, and humans speaking. (A) The different sound frequencies of rain, thunder, and humans speaking were recorded once to be 75 Hz, 125 Hz, and 200 Hz respectively. Playback series were conducted at the Red Cedar River behind Wells Hall at Michigan State University, for one hour, two days a week, for eight weeks. A fifteen second interval began, and one of the three sounds was played from a Bose SoundLink speaker one meter from the edge of the river. The number of ducks performing one of the ten behaviors was recorded. This was repeated for the remaining nine behaviors, and then for the other two environmental sounds. The purpose of the playbacks was to observe how duck behavior changes with increasing sound frequencies. We predict that the ducks will display more alert behaviors in response to humans speaking, but will display the same amount of behaviors in response to one constant frequency, because higher frequencies disturb the ducks' natural environment. (Goudie et al., 2004). The average percentage of ducks performing each of the ten behaviors was calculated by dividing the total amount of ducks performing each behavior divided by the total amount of ducks present. Every percentage found for each trial was then averaged. A chi-squared test was performed to determine if the categorical data of the three environmental sounds was significant. We predict that the p-value will be less than 0.05, making the data significant, because the data is not consistent with the null hypothesis (Sanfilippo et al., 2015). (B) The rain, thunder, and humans speaking sounds were altered to one constant frequency of 300 Hz using Audacity 2.1.2, and a series of playback experiments were conducted using these three sounds. The same methods from part A were used, as well as the same statistical tests. (C) This is an animated gif displaying the ten behavioral responses in ducks. The responses include: agonism, alert, courtship, feeding, locomotion, peering, preening, rest, social, and vigilant.
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Predicted Figure 2. Predicted results of the average percent of humans performing one of ten different behaviors in response to the different frequencies, and one constant frequency, of environmental stimuli: rain, thunder, and humans speaking. (A) The different sound frequencies of rain, thunder, and humans speaking were recorded once to be 75 Hz, 125 Hz, and 200 Hz respectively. Playback series were conducted at the CATA Bus Station located at the center of Michigan State University, for one hour, two days a week, for eight weeks. A fifteen second interval began, and one of the three sounds was played from a Bose SoundLink speaker one meter from the edge of the parking lot. The number of humans performing one of the ten behaviors was recorded. This was repeated for the remaining nine behaviors, and then for the other two environmental sounds. The purpose of the playbacks was to observe how human behavior changes with increasing sound frequencies. We predict that the humans will display more alert behaviors in response to humans speaking, but will display the same amount of behaviors in response to one constant frequency, because higher frequencies disturb the humans' natural environment. (Goudie et al., 2004). The average percentage of humans performing each of the ten behaviors was calculated by dividing the total amount of humans performing each behavior divided by the total amount of humans present. Every percentage found for each trial was then averaged. A chi-squared test was performed to determine if the categorical data of the three environmental sounds was significant. We predict that the p-value will be less than 0.05, making the data significant, because the data is not consistent with the null hypothesis (Sanfilippo et al., 2015). (B) The rain, thunder, and humans speaking sounds were altered to one constant frequency of 300 Hz using Audacity 2.1.2, and a series of playback experiments were conducted using these three sounds. The same methods from part A were used, as well as the same statistical tests. (C) This is an animated gif displaying the ten behavioral responses in humans. The responses include: agonism, alert, courtship, feeding, hygienic, locomotion, peering, rest, social, and vigilant.
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Predicted Figure 3. Predicted results of the average percent of mallard ducks and humans performing one of ten behaviors in response to the control speaker playing no sound. (A) The experiment was carried out by the Red Cedar River behind Wells Hall at Michigan State University, for one hour, two days a week, for eight weeks. Two identical Bose SoundLink speakers were placed at the edge of the river and played no sounds. The speakers with no playbacks acted as a negative control to confirm that the mallard ducks did not react solely to the visualization or location of the speakers. A fifteen second interval began, and the number of ducks performing one of the ten behaviors was recorded. This was repeated for the remaining nine behaviors, and then for the other two environmental sounds. The responses included: agonism, alert, courtship, feeding, locomotion, peering, preening, rest, social, and vigilant. The average percentage of ducks performing each of the ten behaviors was calculated by dividing the total amount of ducks performing each behavior divided by the total amount of ducks present. Every percentage found for each trial was then averaged. We predict that there will be a decreased number of ducks displaying agonism, alert, and vigilant behaviors because those are expected when the ducks are disturbed or agitated by high frequencies of sound (Goudie et al., 2004). However, we predict that there will be an increase in courtship, feeding, locomotion, and rest behaviors because these are natural duck behaviors that they can focus on without the disruptions of rain, thunder, rain or human speaking (Goudie et al., 2004). A chi-squared test will be performed to determine if the categorical data of no sounds was significant. We predict that the p-value will be less than 0.05, making the data significant, because the data is not consistent with the null hypothesis (Sanfilippo et al., 2015). (B) This is a video displaying duck behaviors in their natural environment with no playbacks performed. (C) The experiment was carried out at the CATA Bus Station located at the center of Michigan State University, for one hour, two days a week, for eight weeks. The same methods as part A were used, as well as the same statistical tests. We predict that there will be a decreased number of ducks displaying agonism, alert, and vigilant behaviors because those are expected when the humans are disturbed or agitated by high frequencies of sound (Goudie et al., 2004). However, we predict that there will be an increase in courtship, feeding, locomotion, and rest behaviors because these are natural humans behaviors that they can focus on without the disruptions of rain, thunder, rain or human speaking (Goudie et al., 2004). (D) This is a video displaying human behaviors in their natural environment with no playbacks performed.
Predicted Figure 4. Predicted results of Polymerase Chain Reaction (PCR) products, duck DNA, and human DNA, using gel electrophoresis. The x-axis labels the respective wells while the y-axis is the weight of the DNA particles in base pairs (bp). The weight decreases as the y-axis increases because it is easier for lighter molecules to travel further. For humans, the forward primer was 5'-AAGAGGCAGCGGCTGTG-3' and the reverse primer was 5'-GTAAACCCACGGACCAGAGG-3' (NCBI, 2013). For ducks, the forward primer was 5'-ATCCAACCAGTAGCAAGGCT-3' and the reverse primer was 5'-TAAATCCTCGCACCAGAGGC-3' (NCBI, 2013). Well one consists of the molecular weight ladder Precision Plus Protein Dual Color Standards, well two contains the DNA of the duck blood sample, well three holds the DNA of the human blood sample, and well four contains the molecular weight ladder of water. Well one acts a positive control because it contains the entire genomic sequence which contains the SLC26A5 gene. Well four acts as a negative control because it does not contain any base pairs. The ladder was used to help determine the weight of the DNA in samples, thus determining which genes were located within or missing from the genome. The ladder yielded the same results for every test, so its bands were not predicted. The duck blood was genotyped using the EZ Fast Blood/Cell PCR Genotyping Kit. The DNA was extracted from both the whole duck and human blood. Both were denatured at 95 degrees Celsius for five minutes, and the extension temperature was set at 72 degrees Celsius for ten minutes (Ghatak et al., 2013). The agarose gel concentration was 1%. A red dye was already added to the PCR products due to the 2X PCR Master Mix II used in the DNA extraction sequence. The PCR products were placed under 80 V of current for 30 minutes. We predict that the bands in wells two and three, ducks and humans, will be roughly 2,343 base pairs, because this is the weight of the SLC26A5 gene, and if that molecular weight is found using gel electrophoresis then it must be present in both species (Nikiforov et al., 2012).
Figure 5. Channeling Jane Goodall. This is the five minute documentary video, Channeling Jane Goodall, which encompasses the whole of the experiment for the LB 144 research project.
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