Introduction

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     P. atricapillus, known commonly as black-capped chickadees, are the most geographically widespread type of chickadee in North America. Small, noisy, and non-migratory, these birds can often be seen at feeders at all times of year. Common predators of the chickadee include shrikes, owls, and hawks, as well as tree-climbing mammals. It should be noted, however, that the tree-climbing predators often prey on eggs and hatchlings instead of the full-grown chickadees, while the hawks and owls often eat the adults (National Geographic, 2006).

     The chickadees’ defense from predators come in two main categories: avoidance and reaction. Avoidance of predators is aimed at minimizing the possible number of times that an organism might meet a predator, usually through methods such as camouflage, habitat selection, and limited sociability (Sih, 1982). Reactivity, however, is fully designed to directly influence the outcome of the encounter between the organism and the predator, generally by fighting back through either physical or chemical means (Gottfried, 1979; Ben et.al, 2015). This experiment was focused on the latter of the two categories, reaction.

     Birds react differently to different types of predators, depending on several factors, which include color, size, speed, and rarity in the area (Kruuk, 1964). According to Kruuk, birds have also been known to alter their reactions to predators depending on the perceived threat and intent of the predator (Kruuk, 1964). However, birds will also react to other birds of the same species, presumably due to the desire of each specific organism to pass on its genetic material (Gause, 1932). As a result, birds will often fight each other as well as birds from competing species in order to obtain the best resources.

     Humans, on the other hand, react in a similar fashion. In 2014, a researcher named James Bushman and his colleagues performed an experiment in which they measured glucose levels of married couples. Subjects stuck pins into a voodoo doll that represented their spouse each night, and blasted their spouse’s ears with noise through headphones. The study determined that subjects who had lower glucose levels stuck more pins into the voodoo doll and blasted their spouse with louder and longer noise blasts (Bushman, 2014). From this, it was determined that lower glucose levels, which are correlated to hunger, are also related to higher levels of aggression in humans.

     Previously, a group of researchers led by Timothy Boswell determined in a series of primary and follow up experiments that the expression of a certain protein increased dramatically in broiler chickens that had been fasted for a 24 hour period, as opposed to chickens that had been allowed to feed freely (Boswell et. al, 1999). This protein, known as Neuropeptide Y (or NPY, for short) (HUGO, 2016), is produced by the aptly named NPY gene, located on the long arm of the 7th chromosome in humans. This gene, after its discovery by Morris et. al in 1982, was later linked to survival mechanisms in humans, such as immune response, hunger, and stress response (Goldstone, 2002).

    Since the NPY protein and NPY gene are present in broiler chickens and humans, it was hypothesized that the NPY protein and, by effect, the NPY gene are present in black-capped chickadees, and that they serve similar functions in both humans and birds. Experimentally, it was hypothesized that if there were limited food sources for the black-capped chickadees, then they would behave aggressively due to the increased amounts of NPY protein expression. Similarly, it was hypothesized that humans would behave in the same manner as the black-capped chickadees, where competition for a limited food source would cause humans to behave aggressively. However, it seems that little research has been conducted on whether the functions of the gene and protein are indeed similar in both species. As such, hunger and aggression in both black capped chickadees and humans was observed in this experiment.

Methods

Finalized by A51297226

Chickadee Observation

     Observations were made on black-capped chickadee birds during the months of September through October. Two bird feeders were set up outside of C7 Holmes Hall, disturbed and undisturbed. The undisturbed feeder was set up so that no human contact could occur within fifty feet, while the disturbed feeder could be used for any manipulations needed for the experiment. The undisturbed feeder served as a negative control for human interaction: to make certain that bird behavior was not affected by human presences. The disturbed feeder was manipulated to limit food supply. Of the four feeders that were hung on the feeder, three were taken off to create competition amongst the birds during feeding time.

     To document behavior during the time of the removal of the feeders, video observations were made using the smartphone cameras of two iPhones and one Moto E. The video evidence was then reviewed in slow motion in order to accurately identify aggressive behaviors. Behaviors included wing-flapping, defined as when the bird flapped its wings at or past a 45-degree angle from the resting position in response to another bird; chasing, when a bird flew in towards the feeder and the original occupier of the feeder flew away; and guarding, when a bird perched on the metal crossbar holding the feeders for more than three seconds, and then proceeded to feed directly afterwards.

     Any defined behaviors that were displayed by the chickadee birds were counted as ‘expressed’, while behaviors that were not expressed or within the definition were counted as ‘null’. Any bird that expressed or did not express these behaviors while not in the presence of another bird was not included in the data.

Homologous Behavior

     Similar to chickadee observation, human aggression during feeding time was observed. To create randomization, and to minimize human bias, a random number generator was administered to determine a subject in line at the dining halls. By visiting the Shaw, Brody, and Snyder-Phillips dining halls during popular feeding times, the group was able to observe angry and aggressive behaviors. Defined behaviors included jittering legs, when the subject tapped their foot by continuously lifting the toes or heel of the foot for a period of over five seconds; knee-shaking, defined as movement of the knee parallel to the sagittal plane of the body; arms crossed, defined as when the fingers of the subject were located at or past the pectoral muscle of the body during the arm-crossing; and facial expressions, when the subject wrinkled their brow and/or pursed their lips.

     After the behavioral analysis, a checklist was used to record observations. This checklist was organized to first list the defined behaviors and check off any that were expressed per subject. If the subject did not express the aggressive behaviors, then they were counted as a ‘null’. If the subject did express any of the defined behaviors, then they were counted as a ‘expressed’ in however many categories were observed. After, a survey was given to the previously observed subject (under informed consent) regarding their current levels of hunger and aggression. The survey was written on a 1-10 scale and asked the subject, “How hungry are you?” and “How stressed do you feel right now?” This process was repeated multiple times in order to generate a strong sample size.

Statistical Analysis

     For the statistical analysis of the bird and human data sets, the standard deviation of each data set was calculated in order to determine whether any data was erroneous. The ANOVA unpaired t-test was used for comparing the two data sets, expressed versus unexpressed, indicating the strength of the correlation between the two populations. This test was used due to the fact that we had two or more data sets that were being tested in correlation with one independent variable. The data was then interpreted using the correlational results, the p- and f-values. The error bars for the graphs and the statistical significance of said data sets were calculated using the website VassarStats. This website was used to minimize human error in calculations. Significance was determined based off of the p-value from the ANOVA test. If the p-value was less than .05, then the data was significant.

DNA Sampling and Genetic Isolation

     The protocol for the DNA collection was determined based off of the MSU IACUC Experimental Ethics Standards, as well as commonly used field practices to obtain DNA from birds. It should be noted that the proper documentation of the experimental practices was reviewed and approved by the MSU IACUC. The field protocol was adopted from a Fisheries and Wildlife lab from the Smithsonian (Smithsonian, 2005), and is as follows:

     The birds were captured using a mist net, obtained from a local hunting and fishing goods store. The birds was rendered unconscious through the use of anaesthesia, and a few breast feathers were plucked using forceps. The birds was then released as soon as it woke up, in the area where it was first captured.

    The protocol for the isolation of the DNA was adopted from the protocol created by Bello et. al, and is as follows.

     A 0.5–1-cm section was cut from the terminal portion of the feather quill and placed in a 1.5-ml Eppendorf tube containing 500 ml of lysis buffer (50 mM Tris-HCl, pH 8, 20 mM ethylenediaminetetraacetic acid [EDTA], pH 8, 2% sodium dodecyl sulfate) and 5 µl of proteinase K at a final concentration of 175 mg/ml. The lysis was performed at 37 C overnight with gentle shaking beforehand. After the lysis step, samples were shaken to homogenize the lysate, and then centrifuged at 12,000 g for 10 minutes in order to separate out the supernatant from any undigested soft tissue. The supernatant was then transferred to a clean 1.5-ml Eppendorf tube, labelled with the subject number, and the DNA was purified with 0.5 mL of isopropyl alcohol. Then, the DNA was washed in 0.75 mL of 70% ethanol to precipitate the genomic DNA, and was centrifuged for 2 minutes at 12,000 g. This step was repeated using 100% ethanol. The DNA was then heated at 37 ℃ for 10 minutes, in order to make sure that the remaining alcohol had dissipated from the tube. The DNA pellet was then resuspended in 100–150 ml sterile water.

PCR

     The PCR protocol (standardized for 24 samples of DNA) was adopted from the one used to identify the gene in chickens (Chen et.al, 2007; ThermoFisher, 2016; New England BioLabs, 2016). The primers were synthesized using the Harvard MGH DNA Core Facility. These were the NPY mRNA primers, forward 5'-CGCTGCGACACTACATCAAC-3' and reverse 5'-CTCTGGGCTGGATCGTTTTCC-'3. The DNTPs were ordered from Sigma Aldrich, with product codes D4788 (A), D4913 (C), D5038 (G), and T9656 (T). The Taq polymerase was ordered from ThermoFisher Scientific, using the product code 18038018. The protocol itself is as follows.

     5 µl of 10X Taq, 6 µl of all of the DNTPs, and 4 µl of the forward and reverse primers per sample were all added into one 1.5 mL Eppendorf tube. Sterile water was then added until the volume of the mixture was 50 µl. In the individual 0.5 mL Eppendorf PCR tubes, 1 µl of the master mix, and 1 µl of the specific chickadee subject’s DNA was added. Afterwards, all of the Eppendorf tubes containing master mix and DNA were put into the cycler, and 35 cycles of PCR amplification were run with 45 seconds at 94 ℃ for denaturing; 30 seconds at 55 ℃ for annealing, and 90 seconds at 72 ℃ for extending.

Agarose Gel

     The agarose gel was created using 10g agarose and 90mL of 1X TAE buffer to create a 10% agarose gel. The agarose and 1X TAE buffer were combined in a beaker, and heated until boiling. The resulting liquid was then mixed thoroughly with 5 µl ethidium bromide, and poured into a casting tray.

Electrophoresis

     The agarose gel was placed into an electrophoresis machine, and covered with 1X TAE buffer. Each sample of amplified DNA was then placed into a well in the agarose gel with 1µl of Thermo Scientific 6X DNA Loading Dye. The Kb+ ladder well, which contained 1µl Thermo Scientific GeneRuler 1kb DNA Ladder and 1µl of the previously mentioned loading dye, was used as a positive control. The electrophoresis was then run for 60 minutes at 150 volts.

Results

Finalized by A51303698

Bird Aggression

     The chickadee birds showed angry and aggressive behaviors whilst hungry by demonstrating one or more of the defined behaviors: flapping wings, guarding, or chasing. The behaviors were expressed significantly more when competing for food, indicating aggression (Figure 1). These behaviors occurred because of the competition for food supplies, due to the fact that the number of possible avenues for food were decreased from four to one. With this information, the ANOVA unpaired t-test was used to normalize the data set and to establish how strong the correlation between limited food and aggressive behaviors was. After the correlational data was established and interpreted, the VassarStats website was used to calculate the error bars of the graphs. For all three of the treatment groups, the ANOVA test determined that the f-value was 30.96, while the p-value was 0.039. As 0.039 is less than 0.05, thus the treatment data fell within the standard criteria for significance, in that birds expressed more aggressive behaviors when hungry. However, the p-value for all three of the control groups was greater than 0.05, so the control data did not fall within the standard criteria of significance.

Human Aggression

     Similarly, the humans observed behaved aggressively before eating by expressing one or more of the defined behaviors: jittering legs, crossing arms, and facial expressions in dining halls on the Michigan State campus (Figure 2). However, the results of this observation were inverse to what was predicted; the human subjects displayed small amounts of the defined behaviors. With this information, the ANOVA unpaired t-test was used to normalize the data set and to establish how strong the correlation between the desire for food and aggressive behaviors was. After the correlational data was graphed, the VassarStats website was used to calculate the error bars of the graphs. For the human subject results, the ANOVA test determined that the f-value was 64.51, while the p-value was 0.015. As 0.015 is less than 0.05, thus the treatment data fell within the standard criteria for significance, in that humans do not express aggressive behaviors often when hungry.

PCR

     It was assumed that the products of the PCR would appear as seen in Figure 3, based off of the study by Bello et. al and the Harvard DNA Core website (Bello et. al, 2000; Hyde et. al, 2016), which indicated that any expression of the NPY gene would appear at 253-271 bp and 313-393 bp.

References

Finalized by A51433137

Figures

Finalized by A51303698

Figure 1. A. The number of measured chasing behaviors at the disturbed bird feeder, n= 49. Chasing behaviors, defined as when a bird flew in towards the disturbed feeder and the original occupier of the disturbed feeder flew away, were video recorded during feeding times of the chickadees. The film was then reviewed in slow motion, and the number of expressions (expressed) versus non-expressions (“null”) per group (i.e. 2 or more birds present) were recorded. “Null” was described as a lack of expressed behaviors present, while expressed indicated that the defined behavior was observed. Significance (f=30.96, p=0.039) and error bars were determined by the use of the ANOVA unpaired t-test. B. The number of measured wing-flapping behaviors at the disturbed bird feeder, n=49. Wing-flapping behaviors, defined as when the bird flapped its wings at or past a 45-degree angle from the resting position in response to another bird, were recorded during feeding times of the chickadees. The film was then reviewed in slow motion, and the number of expressions (expressed) versus non-expressions (“null”) per group (i.e. 2 or more birds present) were recorded. “Null” was described as a lack of expressed behaviors present, while expressed indicated that the defined behavior was observed. Significance (f=30.96, p=0.039) and error bars were determined by the use of the ANOVA unpaired t-test. C. The number of measured guarding behaviors at the disturbed bird feeder, n=49. Guarding behaviors, when a bird perched on the metal crossbar holding the feeders for more than three seconds, and then proceeded to feed directly afterwards, were recorded during feeding times of the chickadees. The film was then reviewed in slow motion, and the number of expressions (expressed) versus non-expressions (null) per group (i.e. 2 or more birds present) were recorded. “Null” was described as a lack of expressed behaviors present, while expressed indicated that the defined behavior was observed. Significance (f=30.96, p=0.039) and error bars were determined by the use of the ANOVA unpaired t-test.

A. The number of observed human leg-jittering behaviors, n= 41. Leg-jittering behaviors, as defined as when the subject tapped their foot by continuously lifting the toes or heel of the foot for a period of over five seconds were recorded during the most popular feeding times of the human subjects. Subject behaviors were recorded at either Shaw, Snyder-Phillips, or Brody cafeterias. A random number generator was used to determine which human subject to examine while they were in line. Fourteen people were examined per visit to each cafeteria. If the subject displayed a defined behavior, it was recorded as “expressed”. If the subject did not display a defined behavior, it was recorded as “null”. Significance (f=64.51, p= 0.015) and error bars were determined by the use of the ANOVA unpaired t-test. B. The number of observed human arm-crossing behaviors, n=41. Arm-crossing behaviors, defined as when the fingers of the subject were located at or past the pectoral muscle of the body during the arm-crossing, were recorded during the most popular feeding times of the human subjects. Subject behaviors were recorded at either Shaw, Snyder-Phillips, or Brody cafeterias. A random number generator was used to determine which human subject to examine while they were in line. Fourteen people were examined per visit to each cafeteria. If the subject displayed a defined behavior, it was recorded as “expressed”. If the subject did not display a defined behavior, it was recorded as “null”. Significance (f=64.51, p= 0.015) and error bars were determined by the use of the ANOVA unpaired t-test. C. The number of observed human aggressive facial expressions, n=41. Facial expressions, when the subject wrinkled their brow and pursed their lips, were recorded during the most popular feeding times of the human subjects. Subject behaviors were recorded at either Shaw, Snyder-Phillips, or Brody cafeterias. A random number generator was used to determine which human subject to examine while they were in line. Fourteen people were examined per visit to each cafeteria. If the subject displayed a defined behavior, it was recorded as “expressed”. If the subject did not display a defined behavior, it was recorded as “null”. Significance (f=64.51, p= 0.015) and error bars were determined by the use of the ANOVA unpaired t-test.

Figure 3. Predicted results from amplified products using gel electrophoresis, adapted from Bello, et.al. Thermocycling conditions included a 35 cycles of a 45-second denaturation at 94 ℃, 30 seconds at 55 ℃ for annealing, and 90 seconds at 72 ℃ for extending. The non-discriminating oligonucleotide reverse primer was 5'-CTCTGGGCTGGATCGTTTTCC-'3, and the discriminating oligonucleotide forward primer was 5'-CGCTGCGACACTACATCAAC-3'. The predicted binding site was on the long arm of chromosome 7. It was predicted that no larger DNA fragments would be present in the PCR product, as Taq polymerase helped to mitigate the issue. Lane 1 was predicted to show distinct bands for the target sequence lengths of near 253-271 and 313-393 base pairs.

Fig. 4. Our documentary, Our Lives Among Birds. Data for the video was collected from September 2016 to November 2016, while production was completed near the end of November 2016. Tools used to make the documentary include an iPhone 6, as well as the iMovie program standard to Mac computers.