Discovery of Similar Reactions to Varied Frequencies of Human and Mallards Related to SLC26A5 Gene via Playback Sound
By: B-988, B-887, B-300, B-409
LB 144 Cell and Organismal Biology
Tuesday 1:50pm
Dr. Doug Luckie
11/19/2019
https://msu.edu/~pakrayar/
Finalized By: B-887
Abstract
Authored By: B-887, B-300, B-988, B-409
A comparative research study was conducted between Mallards and Humans at Michigan State University with the purpose of analyzing their responsive behaviors and the possible presence of the SLC26A5 gene within both species. The homologous behavior study was scientifically significant because it provided evidence for similar evolutionary gene development in humans and other species such as Mallards. It was hypothesized that both species will be able to hear and respond in abundance to frequencies known to be well within their hearing range. The cochlear amplifier, a positive feedback mechanism allowed for hearing through the outer hair cells (OHC) in the cochlea (Matsumoto et al, 2010). As vibrations of sound pass through the ear, the hair within is able to absorb and amplify frequencies (Ashmore, 1987). In the experimental design a series of sound playbacks at different frequencies were utilized to exploit the SLC26A5 gene, which is associated with hearing ability. Following field experiments, Polymerase Chain reaction Tests were run in order to isolate target DNA that contains the anticipated nucleotide sequence. Once the PCR was completed, gel electrophoresis was utilized to prove the presence of the gene, SLC26A5 in humans. Prior to experimentation, it was predicted that the gene SLC26A5 is present in both humans and Mallards because they originated from common ancestors (Okoruwa et al, 2008). The gene SLC26A5 should have around 2,000 base pairs (Liu et al, 2003). The study supported the presence of the SLC26A5 gene.
Introduction
Authored By: B-887
Mallard Ducks (Anas platyrhynchos) can be found in or near the Red Cedar River at Michigan State University. Before Mallards migrate for the winter, they build up fat reserves through consumption during their travels South. (Bengtsson et al, 2014). The mating process is imperative for Mallards as females hope to attract a mate before the cold hits, and produce offspring with their spouse. Female Mallards communicate with an inciting call to indicate they are looking for a mate while making sideways head gestures before, during, and after their calls (Abraham, 1974). Following these communications, Female Mallard Ducks then initiate physical interaction by swimming to their desired mate (Abraham, 1974). Furthermore, Mallard Ducks hear and vocalize in a frequency range of 100-4,000 Hz (Abraham, 1974). For mammals, the Mallard Ducks have a narrow hearing range. Humans can hear frequencies from 20 Hz up to 20,000 Hz (Steele, 2003). Both of these hearing ranges are relevant in determining how both Mallard Ducks and humans behave in the presence of sound stimulus.
Much like any species, both humans and Mallard Ducks adhere to certain behavioral patterns of some fashion. Some of the Mallard’s most interesting behaviors are found when studying the common reactions to certain mating calls, communications, or other noises. In relation to Mallards, a study on Veery birds was done to showcase the species song recognition through playbacks of male vocalizations from their own kind (Weary et al, 1991). Daniel Weary and his research team were able to conclude that Veeries recognize their own song playbacks at frequencies near their normal range, as opposed to the extremely altered versions (Weary et al, 1991).
In the campus environment of Michigan State University, Mallard Ducks often obtain food from their interactions with humans visiting their habitat, as feeding is a common pastime for college inhabitants. Residing in locations of high human activity have led the ducks to be tamer and more approachable (Drilling et al, 2002). These ducks on campus have become more accustomed to the sounds of humans and other noises they hear around the Red Cedar River. In a natural secluded setting of an off campus environment where Mallards are heavily hunted, they may be circumspect when encountering other species. This causes them to be more cautious and aware of their surroundings (Drilling et al, 2002). These Mallards may respond to stressful situations in a plethora of ways: aggressive head bobs, threatening the aggressor with an open bill, or even jostling forward with a puffed-out chest (Drilling et al, 2002). Mallard Ducks are comparable to others because most ducks have a similar behavioral response. Human’s behavior; however, is somewhat unique towards each individual. Throughout research there may prove to be a common gene overlap between both ducks and humans (Okoruwa et al, 2008). When trying to analyze the reactions of both parties to certain sound frequencies, the research of the gene could be utilized to explain the similarities in behavior as well as the environment and surroundings.
Genes play a significant role in the lives of Mallards and humans. The gene solute carrier anion transporter family 26, member 5 (SLC26A5) encodes the protein Prestin (Minor et al, 2009). Prestin is a transmembrane protein of the cochlea at the outer hair cells and is required for hearing activity (Minor et al, 2009). SLC26A5 is located at 7q22.1. which indicates that the gene is found on the seventh chromosome on the long arm q at position 22.1. (Liu et al, 2003). SLC26A5 is a voltage dependent motor protein, which plays an important role in the frequency selectivity and sensitivity of organism hearing (Homma et al, 2010). Without the gene, major hearing loss or possibility of deafness may occur (Mutai et al, 2013). Mallards and humans originated from a common ancestor, the diapsid lineage and synapsid lineage respectively. These two types of lineages have functionally similar characteristics, as a result of convergent evolution (Wu & Wang, 2019). This indicates that when notes were played to both Mallards and humans, they reacted similarly to the sound. We predict that both Mallards and humans share the gene, SLC26A5, because they both share a common ancestor, along with the gene playing a significant role in hearing (Okoruwa et al, 2008).
Since Mallards are responsive to vocalizations of their own species, we observed how they respond to different frequencies of musical notes (Abraham, 1974). In order to test this, we conducted a playback experiment of different note frequencies to see the different types of responses that the Mallards had to each note. The different notes/frequencies we included in the playbacks were B0(30Hz), B3(247Hz), B6 (1975Hz), B8 (7902Hz) (Suits, 2008). We hypothesized that if B3 and B6 are played on the speaker, the Mallards would have the largest response to the frequencies of these notes because both B3 and B6 have frequencies within the range that the Mallards can hear. It is hypothesized that humans would have be the most responsive to the 10,000 Hz note because it falls right in the center of the human hearing range (Steele, 2003). Notes within hearing ranges elicit a stronger response than notes with either type of abnormal frequencies, although humans have a very large range of hearing, it may be possible that they pay more attention to other notes (Weary et al, 1991). Both Mallards and humans had responses to the different frequencies of notes and exhibit behaviors that correlate to the predicted function and presence of the SLC26A5 gene (Mutai et al, 2013).
Methods
Authored By: B-300
Observational Playback Study
An observational playback study of Mallard Ducks was conducted at Michigan State University in the Red Cedar River between the backside of Michigan State University Library, and Spartan Stadium within a 100 m distance up and down the river at coordinates of 42°43'47.7"N 84°29'02.2"W. Four different frequencies were played for the Mallards along with a control of no sound where the responses and movements were recorded. Humans had a different set of four frequencies that were played near the rock located next to Michigan State University’s Auditorium at coordinates of 42°43'41.1"N 84°28'39.1"W with their responses recorded. All playbacks and observations for mallards and humans were once a week for each organism (2 total days of experiments per week) for a duration of 1.5 hours between 8:00 am - 5:00 pm for 10 weeks. An iPhone X was used to take pictures and videos of both Mallard and human responses to each frequency treatment. All findings were recorded in a lab notebook dating the time, weather conditions, the location of the observations, how many mallards and humans were present, and the number of responses for each treatment.
Playback Study For Mallards
In order to effectively gauge the effects of frequencies at different pitches on Mallards, a few stipulations were put into place. Throughout the playback observational study, the reactions of each Mallard were recorded and categorized into groups based on the similarities in their reactions. A series of four specific frequencies were played on a UE Megaboom 2 speaker along the Red Cedar River. During each playback of music, observers stood 7m away from the speaker, so that the Mallards would not have been distracted by anything other than the frequency emitted. The frequencies selected were 30Hz, 247Hz, 1975Hz, 7902Hz (B.H. Suits, 2008). These notes were chosen based on the hearing range of the Mallards which resides at 100Hz-4000Hz (Abraham, 1974). Moreover, a control of no sound was observed as well, to consider the normal behaviors of Mallards in an undisturbed environment. The speaker was placed approximately one meter from the edge of the water along the river for each trial up to 100m from the original coordinates. Each frequency was played for a total of 3 trials where a full trial consisted of three repeats of three 1.5 second playback of the note with a 3-second pause between each playback at a total of 10.5 seconds. A female mallard vocalization has a duration of 1.5 seconds, therefore, to replicate the female mallard vocalization each frequency played during experimentation was 1.5 seconds (Abraham, 1974). After every trial, a new one did not start until the Mallards retreated to what resembled their normal unbothered behavior. Following the reset of a trial, the speaker was relocated to a new position along the river in order to incorporate variability in where the sound was being played from. This method supported the specific responses from each specific treatment during the observation from each experiment.
Playback Study For Humans
After playing the different frequencies for Mallards, the experiment was replicated using these methods to observe the behavior of humans. The frequencies from the hidden speaker were played from various locations near The Rock. This location was chosen because it is a highly active point on campus. Observers stood about 20 feet away from the speaker to minimize distraction. Different frequencies of notes, 20Hz, 5000Hz, 10000Hz, and 20000Hz, were played for humans along with a control of no sound to observe the natural behavior of humans when no treatment was played. Experimentation consisted of three trials of a one-minute playback where each frequency which was played intermittently. A new trial did not start until a new set of humans began to enter the vicinity where they could hear the sound. These frequencies were chosen because the hearing range for humans was between 20Hz-20000Hz. Two were in the hearing range and two were at the threshold (Weinberger, 2004). An iPhone XS was used to record human behavior to each treatment and data was recorded for each trial of each experiment.
Data Analysis
The datasets for the experiment were the responses the mallards had to the four frequencies played. The responses were, looked towards the speaker, vocalization towards the speaker, swam away from the speaker, and no response. The data sets for humans were the responses to the four frequencies played during the playback. The responses were, head-turning towards the sound, looking around for the sound, asking what the sound was, and no response. To increase data accuracy, experiments were conducted over a span of ten weeks as there were four treatments and one control. One playback observation is conducted for both mallard and human per week. The strategy allowed more accurate data to be gathered since there was a new set of mallards or humans the second time a previously played treatment was played. After all the experiments were completed the most efficient way to display the data was through bar graphs, video recordings showing the responses to the treatments from either organism, diagrams to explain mechanisms, and calculations of chi-squared and standard deviations to determine the statistical significance. All graphs and charts were created through Microsoft Excel.
Gene Analysis
In order to analyze the genes present, polymerase chain reaction (PCR) testing was conducted for humans using DNA from human cheek cells. PCR was used to determine whether the SLC26A5 gene was present in humans. The forward primer was 5’- TGTGGCCATATATCTCACAGAGCC- 3’ (Liu et al, 2003). The reverse primer 5’- CCCGGGTTCACGCCATTCTC- 3’ was designed by analyzing the DNA sequence of SLC26A5. PCR testing was executed with 1µl of 100µM of each forward and reverse primer, .5 µl of 1x Taq DNA polymerase, 1µl of 10 mM dNTP, 2µl of 50 mM MgCl2, 29.5µl dH2O, 10µl of DNA, and 5µl of 10x PCR buffer at a total volume of 50µl (Tang et al, 2005). The PCR mix was pipetted into a microcentrifuge tube and a centrifuge was used to bring the PCR mix that was adhesive to the sides to the bottom of the microcentrifuge tube. The PCR mix in the microcentrifuge tube was put into a thermal cycler for 1.5 hours. The testing for both humans was done initially at 95°C for three minutes. Testing was repeated for a total of 35 cycles of 30 seconds for each at a denaturation temperature of 95°C to allow DNA to separate into two single strands. The annealing temperature was 50°C for 30 seconds to allow primers to pair up with the single strand template (Stoppler, 2016). The test was concluded at 68°C for a total of five minutes, the final temperature was 12°C. While the PCR mix was in the thermal cycler the gel was made in the meantime. The gel was made by adding 45 ml of water, 0.5 g of agarose, 2.5 ml of TBE into a volumetric flask and was mixed. Next, the volumetric flask was put into a microwave for 30 seconds and was taken out left to cool. Once the gel mix cooled down 5 µl of SYBR dye was added into the volumetric flask and was poured onto the clampted tray containing the well comb. The gel hardened and the well comb was removed, then the gel was placed into the electrophoresis chamber. Gel electrophoresis was used to separate the smaller and larger fragments of DNA. The electrophoresis chamber was filled with lithium borate until it reached the max line to cover the gel. After the PCR was complete, the ladder dye (gold bio 100bp) was put into the first well and 10 µl of the PCR mix and 5 µl of the tracking dye was pipetted into the second, third, and fourth well. The lid was placed on the electrophoresis chamber and the voltage was set to 160 volts. The gel was placed onto a UV transilluminator to show the bands.
Results
Authored By: B-988
In the playback experiment, it was shown that Mallards had a high response rate to sounds within their hearing range. Regarding note B0, no response was observed in great volume as it accounted for 88% (39 out of 44) of the observed behavior among Mallards (Figure 1A). Looking towards the speaker (5 Mallards) and swimming away from the noise (1 Mallard) were recorded in miniscule amounts for this note playback as well; however, those reactions may have been due to regular behavior at the time since they occurred at such a low frequency. During the playback of note B3 (247Hz) approximately 80% (47 out of 59) of responses were Mallards swimming away from the sound (Figure 1A). A portion of Mallards facing away at a relatively mild distance from the speaker, turned around towards the noise (7 Mallards). A select five had no response to this frequency that is in their hearing range (Abraham, 1974). It was noted in observations that most no responses were from subjects at a further distance from the speaker than those who did react. The notes played throughout the duration of experimentation were not commonly heard sounds in the natural environment of Mallard Ducks. Note that B6 had the smallest sample size at 33, of the three current data sets (Figure 1A). There were eight Mallards that ignored the noise, four looked towards the speaker, and twenty-one swam away after sounds were played. Ultimately, Mallard Ducks were more receptive to notes played within the realm of their hearing frequency range. The resulting p-value from chi squared testings was 3.87 * 10-21 indicating statistical significance throughout the collected data.
Humans had the most responses to frequencies of 5,000 Hz and 10,000 Hz due to those being in the middle of their hearing range (Pujol, 2018) (Figure 2A). In the 5,000 Hz playback, twenty-seven humans had no response, thirty-two turned their heads, eleven asked what the noises were, and ten looked around. When testing 10,000 Hz, thirty-one humans had no response, twenty-two humans turned their head two humans asked what the noises were and zero looked around (Figure 2A). The most no responses were in the playback for 20 Hz (19 humans) and 20,000 Hz (14 humans). Observations at the Rock at Michigan State University proved that humans have a selective hearing range and only respond to certain frequencies. While there were the most responses to those frequencies, no response is also considered a response to the different frequencies because it supports that humans cannot hear the sound or the sound did not register or give them a response. Each trial frequency was repeated three times on a given day to get the most accurate data and the most results for each playback. The p-value derived from chi-squared test was 1.94 * 10-16 indicating statistical significance.
After playing each of the various four notes for both humans (20 Hz, 5000 Hz, 10000 Hz, 20000 Hz) and Mallards (30Hz, 247 Hz, 1975 Hz, 7902 Hz) Humans responded to the four notes at a percentage of 50.6% compared to 49.4% response percentage of Mallards, which was a 1.2% increase. Humans had a 34.2% no response compared to 65.8% for Mallards which was 31.6% decrease (Figure 3). There was a larger sample size for Mallards (n=257) to humans (n=178) (Figure 3). Percentages were calculated to determine response compared to no response because it allowed the data to be compared without the error of difference in sample size. Percent was calculated by taking n of the species for either response or no response divided by the total sample size of the same species. According to the data collected humans are more responsive to their four notes compared to Mallards. A chi-squared test was conducted and the derived p-value was 1.94 * 10-16 indicating statistical significance.
After the PCR and gel electrophoresis, we predict that humans and mallard ducks share the gene SLC26A5 because they respond in similar manners to different frequencies (Goudie et al, 2004). Once the PCR and gel electrophoresis were conducted, the GoldBio 100bp ladder was presented with distinguishable bands (Figure 4A). The bands range from 100bp to 1,500bp. The bands in well two and four appeared around 160 base pairs (Figure 4A). This means that the DNA fragment is small in size because the molecules traveled quickly through the matrix in the gel. This showed that the SLC26A5 gene has around 160 base pairs in humans (Figure 4A). The graph indicated that the gene SLC26A5 has 160 base pairs based on the migration distance from the wells (Figure 4B).
References
Authored By: B-887
Abraham, R. L. 1974. Vocalizations of the Mallard (Anas platyrhynchos). Department of Ecology and Behavioral Biology University of Minnesota.
Ashmore, Jonathan Felix 1987. A fast motile response in guinea-pig outer hair cells: the cellular basis of the cochlear amplifier. The Journal of Physiology. 388 (1): 323–347.
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Bengtsson, D., Avril, A., Gunnarsson, G., Elmberg, J., Soderquist, P., Norevik, G., Tolf, C., Safi, K., Fiedler, W., Wikelski, M., Olsen, B., Waldenstrom, J. 2014. Movements, Home-Range Size and Habitat Selection of Mallards during Autumn Migration. PLoS ONE 9(6): e100764.
Drilling, Nancy, Rodger Titman, and Frank McKinney. 2002. Mallard (Anas platyrhynchos), version 2.0. In the Birds of North America (P.G. Rodewald, editor), Cornell Lab of Ornithology, Ithaca, New York, USA.
Liu, X. Z., Ouyang, X. M., Xia, X. J., Zheng, J., Pandya, A., Li, F., and Webb, B. T. 2003. Prestin, a cochlear motor protein, is defective in non-syndromic hearing loss. Human Molecular Genetics, 12(10), 1155-1162.
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Figures
Authored By: B-409
B.
Figure 1. Playback results of reactions from Mallards at different note frequencies, B0 (n=45), B3 (n=59), B6 (n=33), and B8 (n=39). All data collected was from three trials for each respective note and was compiled onto a bar graph based on the type of response. Trials were run at Red Cedar River location between MSU Library and Spartan Stadium once a week. One note was played per day. Each trial lasted for 10.5 seconds, playing the note for three 1.5-second-long pulses with three seconds in between each. The x-axis depicts the types of responses observed during playback. Responses consisted of looking towards the emitted sound, swimming away from the sound, or no apparent response at all. Mallards must have initially been looking away from the speaker then turned around to classify as looking towards. For swimming away classification, Mallards were observed in a still position before playback then commenced swimming after the noise was played. No response was noted when Mallards seemed to continue baseline behaviors. Trials of no notes being played were implemented in order to observe Mallards at their normal behavior. The y-axis indicates the total number of Mallards that responded in a certain manner. The number on top of each bar plot displays the number of ducks in that category as the sample size. The percent error for this experiment has been calculated as shown by the percent error bars in the data set (S.E.=1) using a chi squared significance test to determine a p-value of 3.87 * 10-21, the ’*’ indicates statistical significance. B. Series of notes played during Mallard playback study (In order left to right: B0:30Hz, B3:247Hz, B6:1975Hz, B8:7920Hz).
B.
Figure 2. Observed Human Response to Different Frequencies of Notes. Data was collected to determine the number of human responses to different frequencies played. The four frequencies used including; 20 Hz, 5000 Hz, 10000 Hz, and 20000 Hz. After each frequency was played for one minute, the number of humans that were around the speaker at that time was recorded. Of those around the speaker, the number and type of response at each frequency was determined. All data was taken near The Rock at Michigan State University. The four responses recorded were no response, head turning, asking what the noise was, and looking around for the sound. There were the largest number of no responses due to a number of errors including humans wearing headphones, vehicles passing and other events going on at The Rock. The sample size of each response is noted, and a chi squared test is used to represent the significance of the data. (S.E.=1). P-value = 1.94 * 10-16, the ’*’ indicates statistical significance. B. Series of notes played in human playback experiment(In order left to right: 20Hz, 5,000Hz, 10,000Hz, 20,000Hz).
Figure 3. The percent response comparison of humans and mallards for when the four frequency notes were played for their specific frequency range. Data was collected at the Red Cedar River on Michigan State University’s campus for Mallard Ducks and at The Rock located next to Michigan State University’s Auditorium for the human study. Data was analyzed using percentages because there were not equal sample sizes between both the humans and mallards observed. Percent of responses was graphed against a response or no response for humans and response or no response for Mallard Ducks. Blue bars on the graph represent humans which had playback of frequency notes at 20Hz, 5,000Hz, 10,000Hz, and 20,000Hz. Orange bars on the graph represents mallards that had playback frequency notes of 30Hz, 247Hz, 1975Hz, and 7902Hz. Error bars are at 5%. n equals the sample size. P-value = 1.79 * 10-6, the ’*’ indicates statistical significance.
Figure 4. Results of a Polymerase Chain Reaction (PCR) for humans. A. The gel electrophoresis from the Polymerase Chain Reaction (PCR) shows the base pair numbers on the right side of the figure and the different wells along the top of the figure. The wells in this figure were a Goldbio 100bp Ladder and Human Genome multiple times. The ladder was a standard Goldbio 100bp Ladder band used to identify the size of the DNA. The PCR for Human genome used the forward primer 5’-TGTGGCCATATATCTCACAGAGCC-3’ and the reverse primer, 3’-CCCGGGTTCACGCCATTCTC-5’ (Liu et al, 2003). The beginning denaturing temperature was 95°C and cycled 35 times for 3 minutes each cycle. Then the denaturing temperature again at 95°C for 30 seconds. The annealing temperatures to allow the DNA primers to attach to the template was 50°C. This cycled for 30 seconds and the extending temperature was 68°C for one minute. (Liu et al, 2003). B. From the gel electrophoresis, a graph was made to determine the number of base pairs in the gene. Using a ruler, a common line was made at the top of the wells, and it was measured from the top of the wells to the bottom of the bands in centimeters for both the ladder and the human genome. A graph was made with molecular size (base pairs) versus migration distance in centimeters and plotted to make the equation y=-276.9x+1960.2. The distance from the top of the wells to the bottom of the bands was plugged into the equation to get the total number of base pairs 160.35.
