Observational/Predator Playback Study Finds Homologous Urgency Behavior in Poecile atricapillus and Homo sapiens





















By: Noor Abdallah, Elizabeth Barton, Phoebe Holmes, Megan Wild

















LB 144 Cell and Organismal Biology

Tuesday 7 PM

Hayden Stoub and George Hyde

12/2/2016

https://youtu.be/KuCrrL5AeyA

www.msu.edu/~bartone6

















(Title page written by: A49905810 Revised by: A52206898 Finalized by: A51046558)

Introduction

Written by: Megan Wild

      The Black-capped Chickadee (Poecile atricapillus) is a non-migratory bird that is found in the northern two thirds of the United States and in Canada (Goodwin, 2013). They are a social species that use various calls to communicate within a population (Ficken et al., 1976). The “seet” and “chickadee” calls are commonly used as alarm calls, in order to warn other nearby birds when they sense danger. Danger can come in many different forms for the Black-capped Chickadee. Common predators of this bird includes hawks and owls, as well as smaller mammals that are capable of climbing trees (Templeton, 2005). A threat may come at any time of vulnerability, such as when birds or mammals are present at a bird feeder. While the phonetics of the alarm call is essentially the same, it can change slightly depending on the level of danger that is sensed by the Black-capped Chickadee (Templeton, 2005).

      An observational study was done by Millicent S. Ficken and his group at the University of Wisconsin – Milwaukee Field Station, the purpose for this study was to identify the functions of different Black-capped chickadee calls (Ficken et al., 1978). Fee-bee, gargle, subsong, broken dee, and twitter are a few of the calls that were identified (Ficken et al., 1978). This study concluded that the Black-capped Chickadee had a more elaborate acoustic repertoire than other less social species (Ficken et al., 1978). Studies on the vocalizations of the Black-capped Chickadee have been done in order to observe their response to a threat. One study, by Christopher N. Templeton and his colleagues, observed alarm calls of the Black-capped Chickadee when a predator was detected (Templeton, 2005). In their predator presentation experiments a group of chickadees were exposed to a variety of common predators including 13 live perched raptor species and two live mammalian species of predators (Templeton, 2005). They concluded that the alarm call differed in response to the degree of threat that the predator posed (Templeton, 2005).

      It is hypothesized that the Black-capped Chickadee is able to produce variations in the “chickadee” warning call, because the presence of a gene, such as the FOXP2 gene, which is responsible for speech and language development, allows them to react differently depending on their environment. If a predator is present, it is predicted that the Black-capped Chickadee will vocalize a call with additional “dee’s” if the predator poses a larger threat (Templeton, 2005). For example, if a small group of chickadees are at a bird feeder when a human approaches, one may vocalize a small call to warn other birds of its presence, or even to warn the predator itself. However, when a larger threat, such as an owl, approaches, the call will be longer, to communicate the urgency of the situation (Ficken et al., 1976). Although the “chickadee” song is learned by imitation and used by both males and females, it is expected that the ability to produce variations of it is due to the presence of the FOXP2 gene (Miller et al., 2008). Without the proper presence of this gene, the chickadee would not be able to quickly communicate danger through variations in their call (Miller et al., 2008).

      Similar to Black-capped Chickadees, humans communicate urgency by using variations and inflections in their vocalizations. It is hypothesized that humans are able to produce variations in their speech to communicate the intensity of a situation, due to the proper presence of the FOXP2 gene, allowing surrounding people to understand the urgency of the situation and react accordingly. It is expected that when a human is attempting to communicate urgency to either surrounding people, or even to a threat itself, they will increase the rate of speech as the intensity of the situation increases. Humans contain the FOXP2 gene, a gene found to directly regulate the expression of the CNTNAP2 gene, which relates to speech and language development (Vernes et al., 2008). When a song bird sings, the level of the FOXP2 protein, coded for by the FOXP2 gene, decreases (Miller et al., 2008). This may allow for the expression of other genes, such as the CNTNAP2 gene, therefore producing vocal variability (Miller et al., 2008). If the development of this gene is affected in humans, the person may have speech and language disorders, and won’t be able to communicate properly (Vernes et al., 2008).


Methods

Written by: Phoebe Holmes

Organismal:

      Video and audio recorders were set up 25 feet west of a feeder deemed undisturbed by the Lyman Briggs College faculty, located 40-50 feet from a sidewalk that sees a very low flow of people, next to a wooded, quiet area, just north of Holmes hall. A tape recorder was used to record the vocalizations, and a video recorder was used to capture video data and action shots of the chickadees. The chickadees were observed at both the undisturbed feeder, and also a series of trees/bushes located within a forty foot radius of the feeder. The visual and audio recordings were collected when the chickadees produced vocalizations, and the situations were analyzed to determine the potential meaning behind each specific call. Observations were made for three weeks in intervals of thirty minutes, for 31 consecutive days throughout the month of October. Since Black-capped Chickadees are monomorphic, females and males were not distinguished apart, and data was collected as a whole species (Ficken et al., 1978). In order to eliminate any human bias and increase randomization, a random number generator was used each day to determine the specific time for observation between the hours of 11am and 4pm, the hours that the chickadees were most active. In this way researchers were able to unlock parts of the Black-capped Chickadee’s language and test these parts in the following playback experiments.

      A playback study was then conducted for ten days, over a period of two weeks in November, using the predatory calls of three species of known threats of the chickadees: the great horned owl (Bubo virginianus), the red-tailed hawk (Buteo jamaicensis), and the human (Homo sapiens). The territorial hooting call for the owl, the scream call for the hawk, and a homemade recording of human conversation for humans were played from the speaker. A Cambridge SoundWorks OontZ Angle 3 speaker was placed twenty five feet west of the undisturbed feeder and the volumes of all three calls were standardized to 85-88dB using a Galaxyaudio CM-130 to ensure that the chickadee’s reactions were not affected by the volume of the call, only the threat of the species vocalizing the call. The playback experiment was also conducted at times produced by the random number generator between 11am and 4pm. All three predatory vocalizations were played each day for two minutes per threat, with ten minutes of silence between species. A negative control was also added and performed at the beginning of every session, involving ten minutes with the speaker in position producing no sound, with observers present.

Analysis:

      The chickadees’ vocal responses were recording using the tape recorder placed next to the speaker. To analyze calls, the number of “dee” syllables were counted and averaged for each threat. After analyzing the data from the playback experiment and determining the “dee” syllables for each call, the results were compared for each threat. To do this, an analysis of variance (ANOVA) test was performed. In the ANOVA test, the three threats, along with the negative control, were compared to each other in order to determine any statistical significance between populations. These tests were completed using a website for statistical computation called Vassarstats (Lowry, 2016).

Human:

      To further understand the behavior of Black-capped Chickadees, an observational study was conducted with humans as the subjects. The setting was among the various places on the Michigan State University campus: Holmes Hall, The Vista at Shaw, and the Chemistry building. Observations were made of vocalizations when a person expressed one of eight universal facial expressions, as identified by Paul Ekman and his colleagues (Breedlove and Watson, 2013). These facial expressions were defined as: happiness, surprise, anger, sadness, fear, disgust, embarrassment, and contempt. For our observations the emotion was first identified then the level of low, medium, or high intensity was recorded. The seven emotions that were identified along with the corresponding levels were: happy, contempt, joy, sadness, disgust, rage, and loathing (Breedlove and Watson, 2013). A random number generator was used to determine times of observation between the hours of operation for MSU cafeterias (7am-12am), and at which of the three locations. A spinner was used to identify which person in the room would be observed. For each random vocalization recorded, the level of intensity that correlated with the emotion shown was documented as well. In addition, the number of words per minute spoken was calculated. After nine days and nine recordings, the number of words per minute for each intensity level were averaged.

Genetics:

      To further enhance the understanding of the the behavior of both species, and compare the behaviors, the research team investigated at a genetic level. After obtaining DNA samples from researchers, which has been discussed in the LB 144 Course pack, several steps were conducted to isolate the FOXP2 and CNTNAP2 genes. The first step in isolating DNA is DNA purification. Purification steps were followed from the methods described in published research by LaMontagne and colleagues (LaMontagne et al., 2002). Once the DNA was purified, Polymerase Chain Reaction (PCR) tests were used to analyze the purified DNA. A study by Vernes research group isolated the FOX2P gene, so a similar methodology was conducted. In this study the FOXP2 gene in humans was identified as forward primer 5’-CCTTCAGCGTCAGGGACTCA-3’ and reverse primer 3’-CACTTCTTTCCATAACTGCTGAATCTC-5’ (Anonymous, 2016). The FOXP2 and CNTNAP2 primers for Black-capped Chickadees could not be identified, so the primers for the zebra finch, another species of songbird, were used. FOXP2 was identified with forward primer 5’-ATGATGCAGGAATCTGCGACA-3’ and reverse primer 3’-GAAATAGACTTCTAGACCTTACT-5’ (Haesler et al., 2004). The CNTNAP2 primers for zebra finches were identified as forward primer 5’-TGTATCTTTAACTCGTACTGGAGCA-3’ and reverse primer 3’-ACTATTTATTTCTCCTCCCCC-5’ (Anonymous, 2016). CNTNAP2 gene in humans was identified as the forward primer 5’-TCCCTCCACGTCCCAAAAATG-3’ and the reverse primer 3’-TCTTGGCATAGCCGGGAGAA-5’ (Vernes et al., 2008). A PCR test is a 3 step process: denaturation, annealing, and extension. Methods were performed in accordance to those published by Steve Palumbi and colleagues (Palumbi et al., 2002). Denaturation occurred at 94 degrees celsius for 30 (or less) seconds, and annealing occurred at about 55 degrees celsius for 30 - 60 seconds, although temperatures as low as 37 degrees celsius have shown to work as well, and extension could occur at temperatures between 72-75 degrees celsius (Palumbi et al., 2002). This refers to one cycle to gain sufficient sample. The cycle was done 30 times, as directed by the methods stated by Vernes group (Vernes et al., 2008).

      To make the agarose gel for gel electrophoresis, 0.5 grams of agarose was measured. This was then mixed with 5 milliliters of 10X TBE solution and 45 milliliters of deionized water. Then, the mixture was microwaved in 30 second intervals for 1 minute and 30 seconds, or until agarose powder was completely dissolved. The mixture was removed, using gloves, in between intervals and stirred. After all of the agarose powder was dissolved, the solution was left to cool for 5 minutes. Next, 5 micrometers of SYBR was added to the solution. The solution was then poured into the gel tray and left to cool, with the comb in place. Once the gel was hardened, the comb was removed and samples of DNA were pipetted into the wells. The lid was then placed and the electrodes were connected. The gel was then run at 100V for 30 minutes. At completion, the gel was analyzed over ultraviolet light to visualize the bands to determine base pairs.

Results

Written by: Noor Abdallah

      Black-capped Chickadees have a variety of calls in their vocal repertoire, and previous research has found that these calls include a gargle, broken dee, begging dee, chick-a-dee call complex, faint feebee, and a fee-bee call, among many others (Ficken et al., 1976). The “fee-bee” call is only given by males, and is thought to be a declaration of territory, used most frequently between April and July (Ficken et al., 1976). The “chick-a-dee” call has been found to be a warning call, used when a threat is present, to coordinate group movements (Ficken et al., 1976). In the observational study, one “chick-a-dee” call was identified and found to contain three “dee” syllables (Figure 1). It was documented when the feeder was approached by a human threat, and an ANOVA test was performed to determine a variance of 0 (Lowery, 2016). When threatened by a squirrel, several chickadee calls were heard, with an average of 3.5 “dees” and a variance of 0.70 (Lowery, 2016). When threatened by others of the same species (other Black-capped Chickadees), the average number of “dees” was calculated to be 2.5 with a variance of 4.8 (Lowery, 2016). When no other animals were observed, the average number of “dees” was found to be 3.5, with the variance being calculated to 0.980 (Lowery, 2016). The duration of the call has been found to correlate to the size of the threat an animal poses (Templeton, 2005). However, the variance statistics do not portray such results due to the uncertainty of the predator approaching. Although a predator was observed and an alarm call vocalized, it is uncertain that the chickadee was responding to the observed species. In addition, the “chickadee” calls recorded with no other birds present may have been in response to a stimulus not observed by the researchers.

      The playback study observations provided evidence that the Black-capped Chickadee’s alarm call will be longer in duration when a larger threat is present, as previous research also shows, the urgency in the chickadee’s warning is conveyed through variations in the call (Templeton, 2005). We expect the “chickadee” call to include more “dees” at the end of the call to communicate that a larger threat is near, and less “dees” when a smaller threat is present, therefore altering the length, in seconds, of the call (Figure 2). The average call of the largest threat, the Great Horned Owl is 4.3 shown in Figure 2. An ANOVA tested calculated on Vassarstats shows a variance of 0.20, indicating very little variance. The Red-tailed hawk call instigates an average call of 3 (Figure 2) , showing a variance of 0. The average alarm call for humans was 2.6 with a variance of 0.3. The little variance in the playback study supports our hypothesis that black capped chickadee’s alarm call is distinct for a predator’s level of threat imposed on the chickadees.

      The emotions identified according to their associated intensity levels, using Paul Ekman and colleagues universal facial expressions and levels of intensity, were: happy, contempt, joy, sadness, disgust, rage, and loathing (Breedlove and Watson, 462). The researchers observed that as emotion intensity increased, the average number of words per minute decreased (Figure 3). Positive emotions such as joy, happiness and contempt compared to negative emotions such as rage and disgusted produced a higher average words per minute (Figure 3). Varying forms of communication of the Black-capped Chickadee is compared to the human’s varying forms of communication expressing emotions through speech, both species possess similar communication behaviors. Our observations of both species lead us to further investigate similarities leading the team to the FOXP2 gene in both humans and Black-capped chickadees.

      The research group was able to identify the FOXP2 gene and a related CNTNAP2 gene to be present in both species. Any mutations with the FOXP2 gene allowed for a variance in the amount of the CNTNAP2 gene expressed (Vernes et al., 2008). We predict the Polymerase Chain Reaction (PCR) test will show the FOXP2 gene at a range between 80 and 100 base pairs as shown in Figure 4. We predict that the PCR test will show the CNTNAP2 gene at the range 140- 200 base pairs, shown in Figure 4 as well.

References

Written by: Megan Wild

Anonymous. 2016. NCBI Web Site. https://www.ncbi.nlm.nih.gov/nuccore/NM_014141, last accessed 11/18/16.

Breedlove, S.M., and N.V. Watson. Biological Psychology: An Introduction to Behavioral and Cognitive Neuroscience. Sunderland, MA: Sinauer Associates, 2013. Print.

Ficken, M.S., R.W. Ficken and S.R. Witkin. 1978. Vocal Repertoire of the Black-capped Chickadee. The Auk 95.1: 34-48.

Goodwin, Sarah E., and Jeffrey Podos. "Shift of Song Frequencies in Response to Masking Tones." Animal Behaviour 85, no. 2 (2013): 435-40.

Haesler, S., K. Wada, A. Nshdejan, E.E. Morrisey, T. Lints, E.D. Jarvis, and C. Scharff. 2004. FoxP2 Expression in Avian Vocal Learners and Non-Learners. The Journal of Neuroscience 24(13): 3164-3175.

LaMontagne, M.G., F.C. Michel, P.A. Holden, and C.A. Reddy. 2002. Evaluation of Extraction and Purification Methods for Obtaining PCR-amplifiable DNA from Compost for Microbial Community

      Analysis. Journal of Microbiological Methods 49: 255-264.

Lowry, R. 2016. Vassarstats Web Site. http://vassarstats.net/, last accessed 10/17/16

Miller, J.E., E. Spiteri, M. Condro, R. DosumuJohnson, D. Geschwind, and S. White. 2008. Birdsong Decreases Protien Levels of FOXP2, a Molecule Required for Human Speech. 100(4): 2015-2025.

Palumbi, S., A. Martin, S. Romano, W.O. McMillan, L. Stice, and G. Grabowski. 2002. The Fool’s Guide to PCR. Department of Zoology and Andrew Martin Kewalo Marine Laboratory: 4-26.

Templeton, C.N. Allometry of Alarm Calls: Black-capped Chickadees Encode Information About Predator Size. Science 308.5730: 1934-1937.

Vernes, et al. 2008. A Functional Genetic Link Between Distinct Developmental Language Disorders. New England Journal of Medicine 359: 2337-2345.

Weisman, R. and L. Ratcliffe. 2004. Relative Pitch and the Song of Black-Capped Chickadees. American Scientist 92: 532-539.

Figures

Written by: Elizabeth Barton
Figure 1

      Figure 1. Duration of call with predator present in observational study. The Black-capped Chickadee was observed at the undisturbed feeder outside of Holmes Hall. The behaviors of the bird, as well as the length of the calls were documented in order to identify the different calls. The calls were then grouped together based on the stimuli that were present at the bird feeder. These stimuli were when a human was present, when a squirrel was present, when a bird of the same species was present, when there were no other birds present, and when there were birds of different species present. A bird of the same species would be considered another Black-capped Chickadee and a bird of a different species would be considered any other type of bird that was present at the bird feeder. The calls were then analyzed to determine if there was a difference among the length of the calls depending on the different stimuli that were present at the bird feeder. It was determined that there was some variance among the duration of the calls depending on the type of stimulus present at the bird feeder.



Figure 2a

      Figure 2. Duration of call in relation to predator vocalization played in playback study. The sounds of three different predators were played at the undisturbed bird feeder behind Holmes Hall. The Great Horned Owl, Red-tailed Hawk, and human were chosen as potential predators because the level of relative threat to the Black-capped Chickadee varies among the different predators. The duration of the calls of the Black-capped Chickadee were recorded in response to the predator vocalization and later analyzed to determine the average length of the call. The length of the call was determined by recording the number of “dee” notes in the call made by the chickadee. This number was then averaged to determine the average length of the call, in relation to the predator vocalization. It was found that as the relative threat of the predator increased, the length of the call also increased.

Figure 3

      Figure 3. Average words per minute spoken in relation to intensity of emotion expressed. Humans were observed in random locations around campus at various times. The messages that they were communicating were recorded using a tape recorder. The various emotions expressed while the human was speaking were also documented. The emotions were then grouped together depending on their intensity, using the guidelines presented in Biological Psychology by Breedlove and Watson. As the intensity of the emotion that was expressed increased, it was assumed that the urgency of the message that was communicated also increased. The recordings were then analyzed to determine the average words per minute spoken, in relation to the intensity of the emotion. As the intensity of emotion increased, the average words per minute that were spoken decreased. Therefore, as the urgency of the communicated message increased, the average words per minute spoken decreased.

Figure 4

      Figure 4. Predicted results using gel electrophoresis. Polymerase Chain Reaction (PCR) will be used to amplify a DNA segment on chromosome 7, where the FOXP2 gene is located. It will also be used to amplify the CNTNAP2 gene on chromosome 7. The DNA will denatured at 94 degrees celsius for 30 seconds. The annealing temperature will be set at 55 degrees celsius for 30-50 seconds. Then, the extension temperature will be set between 72-75 degrees celsius. The process will be repeated 30 times. The predicted bands will appear between 80 and 100 base pairs for the FOXP2 gene and between 140 and 200 base pairs.

      Figure 5.