Written by: Chiara Bowen, Annelise Kulpanowski, Megan Mulheron

Revised by: Megan Mulheron

Finalized by: Annelise Kulpanowski

         The transferring of genetic information from parent to offspring, often referred to as the preserving effect, allows for key traits and characteristics to remain apparent in a species throughout the passing of time (Advantages and Disadvantages of Sexual Reproduction, 2014). In order to retain genetic information from one generation to the next, the Mallard duck uses sexual intercourse to pass DNA from parent to offspring (Kalita, Behavioral characteristics of domestic ducks). It is this reproductive process of the Mallard duck that has been observed to take place throughout the months of September to December, often in bodies of freshwater spanning across the Northern hemisphere, specifically North America, Europe, and Asia (Hohn, 1947). The reproduction that occurs throughout the summer into the winter involves key display responses that the male Mallard duck uses to attempt to attract females (Johnsgard, 1961). While the anatomical and seasonal preferences for human intercourse is much different from that of the Mallard duck, it too is thought that males demonstrate specific display responses in order to try attract a mate, just as the male Mallard ducks do (Moore, 1995).

         In terms of Mallard duck mating, a male exhibits 3 characteristic displays in order to try and court a female: head-up tail-up, head-bobbing, and the grunt whistle (Barry, 2015). The Head-Up Tail-Up display is often observed when a male stretches his wings outwards and exposes his secondary feathers underneath (Barry, 2015). This behavior is typically accompanied by a loud whistle (Barry, 2015). The head-bobbing gesture is observed when the male Mallard duck bobs his head up and down repeatedly in front of a female over the period of a few seconds (Barry, 2015). The grunt whistle is a very quick gesture observed when a male Mallard duck raises its head out of the water and emits a distinct whistle, which is typically followed by a grunt like noise (Barry, 2015). Paul A. Johnsgard in his study, focused on the grunt whistle and head-up tail-up gestures and determined that the grunt whistle was performed more commonly than the head-up tail-up due to being a simpler display (Johnsgard, 1961). Male humans have many different behaviors in order to attract a female as well. Three common behaviors exhibited by a male that elicit a positive response from a female are: arm flexion, holding a females hand, and primping (Moore, 1995). Arm flexion is used in order to demonstrate dominance and strength. Holding a females hand demonstrates interest, and lastly, primping is used to before or during engagement with a female in order to get a male to appear more attractive. Depending on how these males conduct these specific mating gestures, the females will respond by either accepting them or rejecting them and looking for a new partner (Holmberg, 1989).

         The shared instinctive attraction techniques found in both the male mallard ducks and male humans could possibly be attributed to the fact that Mallards are among the 3% of birds with exterior sex organs (Schloss, 2014). This illustrates the fact that there may be genetic similarities between the two. The DRD4 gene, also known as a dopamine receptor, has been linked to both sexual desire and arousal in humans (Garcia et al., 2010). Dopamine is a chemical associated with the body's 'pleasure system' and scientists are aware that sexual behavior in both humans and animals can be controlled by this neurotransmitter (Garcia et al., 2010). A study conducted at Binghamton University demonstrated how dopamine circuits have been proven to create a drive for things including sex, sometimes leading to infidelity and sexual promiscuity (Garcia et al., 2010). This study produced information valuable as to how the DRD4 gene influences human behavior (Garcia et al., 2010). This gene has additionally been tested in rats with findings that emphasized the fact that the DRD4 agonist induced penile erection and significantly associated with desire, function and arousal (Zion et al., 2006). When it comes to the attraction techniques between ducks and humans, both exhibit behaviors that lead to the belief that the species desire sexual interactions and become aroused when attempting to captivate a mate (Moore, 1995). It is thus anticipated that this gene is responsible for the similarities found between humans and ducks in regards to mating behaviors (Zion et al., 2006). In this observational study we will be inspecting male Mallard ducks sexual behavior at the Red Cedar River on Michigan State University's Campus as well as male human sexual behavior at the university cafeterias. We will be examining their behavior and documenting the different techniques used to engage females. It is hypothesized that if the male Mallard ducks and male humans exhibit similar behaviors and gestures when attempting to draw in a mate, then the DRD4 gene is instituted in both species and plays the same role regarding sexual arousal and behavior.

Written by: Chiara Bowen, Annelise Kulpanowski, Megan Mulheron

Revised by: Chiara Bowen, Annelise Kulpanowski

Finalized by: Chiara Bowen

         Observations of both male Mallard ducks and male humans were made over a two month span starting on September 26th, 2016 and ending on November 13th, 2016. Mallard ducks were observed in an urban setting at the Red Cedar River on Michigan State University's campus in East Lansing, Michigan. The chosen location for observation is directly across from the library on campus where ducks are known to be most active during the day. The male mallard ducks were observed once a week on Monday's between 10:00 A.M. to 2:00 P.M. because it is the middle of the day. Male humans were inspected at cafeterias and coffee shops on Michigan State University's campus, which is a location with a high number of men and women leading to sexual tension. This was done once a week at Holmes Hall cafeteria on Wednesdays at 1:00 P.M. to 2:00 P.M. and nearby coffee shops. Both male Mallard's and male humans were analyzed through human observation as well as through video recordings taken using an iPhone 7. All findings were then recorded in a lab notebook using a graphite pencil or ink pen noting the date, time, weather conditions and location of the observations.

         At both the Red Cedar River and Michigan State University Holmes Hall cafeteria, the mating behavior of male Mallard ducks and male humans were recorded respectively. Throughout this time, the male Mallard duck's three specific mating gestures: Head-bobbing, Head-Up Tail-Up, and the Grunt Whistle, were recorded at the location across from the Michigan State library on the Red Cedar River (Johnsgard, 1961). Specifically, the time, place, temperature, male gesture, and female response were recorded in a laboratory notebook with a pencil as the writing utensil (Johnsgard, 1961). The total number of each of the three individual mating displays observed were tallied and counted individually, so if the male displayed head-up tail-up then engaged in nod swimming directly after, the two gestures were tallied exclusively. Various observations from the hour long time period were captured through the use of iPhone 7 video recordings to allow for a deeper analysis to see if any gestures were missed. Additionally, female responses to the male gestures including; nod-swimming, head bobbing, or no response were counted and evaluated (Holmberg, Edsman and Klint, 1989). In order to be considered a response, the female had to perform one of the previously mentioned displays within 5 seconds after the male performed his courtship gesture.

         In order to observe the mating behavior of the human males, heterosexual encounters on campus were the focal point. This is because it is easier to tell that they are using mating behaviors if they are doing these gestures in front of or towards the opposite sex. Three specific behaviors of males were noted, including: arm flexion, holding a females hand, and primping themselves before or during engagement with a female (Moore, 1995). Males demonstrating these behaviors were all observed and videotaped on an iPhone 7. The observations were noted in a laboratory notebook using pencil or pen, where the time, place, temperature, male gesture, and female response were recorded as they occurred. Female responses were recorded in a laboratory notebook based on whether they smiled/laughed, held the guys hand, or showed no response and/or rejected the gesture (Moore, 1995). A smile was defined as the corners of the female's mouth turned up and the front teeth became exposed. Rejection is designated as the female walking away from the male or disengaging in conversation (Moore, 1995).

         Playbacks of both Mallard ducks and humans were done at the same times and places as the observations were. Female Mallard ducks and female humans were analyzed for the types of responses shown after the sounds were played. There were two sounds per species along with no sound. The two mallard duck sounds included a feeding call and the grunt whistle. The feeding call is the positive control while no sound is the negative control. The two sounds for the humans are screams and cat calling. The positive control for the humans is the screaming while the negative is no sound. The sounds were played using an iPhone 7 and a HMDX Hangtime portable bluetooth speaker. The speaker when used for the Mallard ducks was placed on the stone steps or rocks so the ducks could not see it. For the humans it was placed at a table in a way that no one could see it. The sounds were played after the females backs were turned so that if they turned to look at the speaker, we would know they were responding to the sound. The two behaviors looked for in the female was no response or a behavioral response. A behavioral response includes anything from going to look at the speaker to one of the mating behaviors. In order to be considered a response, the female had to perform one of the displays within 5 seconds after the sound was played.All findings were then recorded in a lab notebook using a graphite pencil or ink pen noting the date, time, weather conditions, location, male sound and female response to the playback.

         The findings from the observations and playback were then used to determine the prevalence of the DRD4 gene in Mallard ducks and its potential link to human mating behavior. This was done by using the Bio-Rad T100 Thermal Cycler on Mallard duck feathers and human hair. The feathers came from the ducks that were being studied by the ducks plucking their feathers and the feathers going downstream in the river and the human hair from the heads of the scientists by the scientists plucking their hair out to use for the experiment. There were three steps to finding the DRD4 gene, DNA extraction, Polymerase Chain Reaction (PCR), and Gel Electrophoresis.

DNA Extraction

         The DNA was isolated from hair shafts using microscopic glass-grinding and organic solvent extraction (Ghatak, Muthukumaran and Nachimuthu, 2013). First, a digestion buffer was added to a 1.5-mL microcentrifuge tube, along with 40 µL of 1 M DTT and 15 µL of 10 mg/mL proteinase K because it is a strong reducing agent with relatively high salt content and also an anionic detergent which will decrease the chance of contamination(Ghatak, Muthukumaran and Nachimuthu, 2013). The hair sample was added to this solution before vortexing and incubating for 2 hours at 56°C (Ghatak, Muthukumaran and Nachimuthu, 2013). After 2 hours of incubation, the sample tube was vortexed again, and an additional 40 µL of 1 M DTT and 15 µL of 10 mg/mL proteinase K were added, followed by gentle mixing and incubation at 60°C for 2 hours or until hair was dissolved completely so that the DNA could be extracted from each sample with an equal volume of phenol:chloroform: isoamyl alcohol solution and mixed gently by inverting the tube for a few minutes (Ghatak, Muthukumaran and Nachimuthu, 2013). The samples were centrifuged for 10 min, followed by transferring the upper aqueous layer into a microcentrifuge tube. This is because the upper aqueous layer is where the DNA ended up after being separated by density in the centrifuge. An equal volume of chloroform:isoamyl alcohol was added, and the tube was centrifuged again for 10 min (Ghatak, Muthukumaran and Nachimuthu, 2013). The upper aqueous layer was transferred into a microcentrifuge tube before double the volume of chilled isopropanol and one-tenth volume of 3 M sodium acetate were added. The sample was chilled on ice for 1 hour for the DNA precipitation to occur (Ghatak, Muthukumaran and Nachimuthu, 2013). The sample was then centrifuged for 10 min. The film at the top of the tube was discarded, 250 µL 70% ethanol was added and centrifuged for 10 min. The film was discarded again and was air-dried in a laminar air flow (Ghatak, Muthukumaran and Nachimuthu, 2013).

PCR

         To analyze the DNA obtained from the extraction process, the combination of 2.0 µL of the target DNA template obtained through DNA purification, 5.0 µL 10X PCR buffer, 0.2 µL Taq polymerase because it copies the DNA sequence, 2.0 µL primer which was purchased on OriGene with a sequence of 5'- AUG CUG CUG CUC UAC UGG -3', 1.0 µL 10 mM deoxynucleotide building blocks (dNTP) of DNA and 40.4 µL water was added to a test tube on ice (Elnifro, et al., 2000). The combination was mixed and spun down in a centrifuge. Once the sample was placed in the Bio-Rad T100 Thermal Cycler, it went through three steps: initial denaturation, primer annealing, and extension. In the initial denaturation stage the temperature was raised to 94°C which disrupts the hydrogen bonds between complementary bases and the reaction is incubated for 2-5 min to ensure that all complex, double-stranded DNA molecules are separated into single strands for amplification (Sigma-Aldrich, 2016). In primer annealing, the temperature was lowered to approximately 5°C below the melting temperature of the primers, 45-60°C, to promote primer binding to the template. In extension, the temperature was increased to 72°C, which is optimum for DNA polymerase activity to allow the hybridized primers to be extended (Sigma-Aldrich, 2016).

Gel Electrophoresis

         0.4g of agarose was put into a flask with 4 mL of 10x TBE and 36 mL of water. The solution was then put into a microwave for 10 sec increments until agarose was melted. It had to then be cooled before the 4 µL of 10,000x SYBR dye was added otherwise the dye would not work as well (Wahl et al., 1979). The gel was then poured into the gel mold with a comb on one end. (Genetic Science Learning Center, 2013). A buffer solution of 1x TBE was then poured into the electrophoresis box after the gel was hard. Lanes 1 and 5 were pipetted with the positive control, samples of DNA amplified by the control primers (Galdzicki and Haradon, 2014). DNA samples from one scientist using primer set 1 and primer set 2 were pipetted into lanes 2 and 3 (Galdzicki and Haradon, 2014). Lane 4 was pipetted with the negative control, no DNA. DNA samples from the other scientist, using primer set 1 and primer set 2 were pipetted into lanes 6 and 7 (Galdzicki and Haradon, 2014). Pipetted into Lane 8 was the DNA ladder, a standard set of DNA lengths (Galdzicki and Haradon, 2014). The gel ran and then was put on an ultraviolet light (Genetic Science Learning Center, 2013). The lengths of the DNA were then determined with base pair length (bp) from the positive control. The DRD4 gene is approximately 3,414 bp (Weizmann Institute of Science, 2008).

Results

Written by: Chiara Bowen, Annelise Kulpanowski

Finalized by: Annelise Kulpanowski

         We predict that the display performed most frequently by male Mallard ducks will be the grunt whistle (Figure 1A), because as shown in Paul A. Johnsgard's study, 46% of the displays performed by Mallards were grunt whistles (Johnsgard, 1961). The grunt whistle is used to impress the females and it is low intensity. This shows that it would require the least amount of energy and therefore will be used most to attract the females. We predict that males with a high display activity are more likely to be incited by females (Figure 1B), because in the study Female Mate Preferences and Male Attributes in Mallard Ducks, it was found that the more active the male, the more females there were attracted to it (Holmberg, Edsman and Klint, 1989). The more active the male is, the more likely the female is to respond or take notice. Since these gestures involve movement and sound, they will catch the female's eyes and ears, encouraging them to respond. If the males do not do many gesture it will be less likely they will attract a female as a result, the more active the male the more successful he will be in attracting a female for a mate.

         We predict that primping will be the most common courtship gesture performed by male humans (Figure 2A), because as shown in Monica Moore's study, approximately 55% of the gestures performed by females was primping and we expect the results to be similar for males (Moore, 1995). Based on prior unnoted observations and knowledge, primping is a very common way for both males and females to prepare themselves for an exchange with the opposite gender. We predict that when a male performs a hand holding gesture, females will respond with a hand hold more than 70% of the time (Figure 2B), because David Buss, in his study, found it to be one of the most effective techniques performed by males when attempting to attract a female (Buss, 1988). Often times, males will only hold a females hand if he is confident that she will hold his hand back due to fear of rejection. For that reason, we prepare to see many reciprocated hand holdings compared to rejections. We predict that for both arm flexion and primping, females will show no response greater than 50% of the time (Figure 2B), because it was found that these gestures are used more to impress rather than to elicit a response (Moore, 1995). Arm flexion and primping are both very common among the male species which overtime has led it to become unnoticed by most females. Females often just ignore males attempts to show off or look good in front of them due to habituation, so we expect to observe many cases of male arm flexion that results in no response from a female.

         We predict that the no sound speaker will result in minimal behavioral response from female Mallard ducks and humans (Figure 3A and Figure 3B), because Ulagaraj and Walker in their similar experimental design found minimal response from mole crickets using a no sound control (Ulagaraj and Walker, 1979). The no sound is a negative control meaning we expect no response to come from it. We predict that the feeding call will result in a maximal behavioral response from the female Mallard ducks (Figure 3A), because Bretagnolle in his study found that the use of a known call elicited a response by the storm petrels (Bretagnolle, 1989). The feeding call is a positive control in this experiment and is expected to evoke a response from the female Mallards. We expect similar results for the scream in the human study which is also used as a positive control. We predict that the grunt whistle will trigger a female response 30% of the time (Figure 3A), because as found in Tuttle and Ryan's study species rely on both visual and auditory cues (Tuttle and Ryan, 1981). We expect that without both the visual and auditory cues the female Mallard ducks are not very likely to show a behavioral response.

         We predict that the results of our gel electrophoresis will show that the DRD4 gene is present in the Mallard duck genome as well as humans, because Zion and his colleagues found that most mammals including humans and mice have the gene present in their genome (Zion et al., 2006). We predict that the DRD4 gene will appear as shown in lanes 2 and 6 in gel electrophoresis (Figure 4), because Goedecke and his colleagues tested the DRD4 gene and the findings were as shown in those lanes (Goedecke et al., 2009). We predict that lane 4, the negative control lane, will show no sign of DNA (Figure 4), because Galdzicki and Haradon in their gel electrophoresis used a negative control similar to ours which resulted in the appearance of no DNA (Galdzicki and Haradon, 2014). We predict that lane 8, the positive control, will appear very bright and abundant (Figure 4), because Galdzicki and Haradon in their gel electrophoresis used a DNA positive control with similar results (Galdzicki and Haradon, 2014). We predict that the Mallard duck DNA when combined with a dye that would react with UV light will present a glow of RNA around 3,414 base pairs (Figure 4), because of the data found on the Human Gene Database (Weizmann Institute of Science, 2008).