Introduction
Finalized by: Olivia Bayer
Social hierarchies are how individuals interact with each other to form a linear ladder. This ladder is structured with dominant individuals on top and subordinates on the bottom. Social, or dominant, hierarchies can be found in many communal-living species in nature such as birds, fish, and mammals- including humans (Chase et al, 2002). In this study, a Nano community comprised of four species of fish, Hatchet, lamb chop rasbora, dawn tetra, and blue ram cichlid, will be observed to determine if a social hierarchy exists between the different species. Obtaining a dominant social status can lead to many benefits such as greater access to food, shelter, and sexual partners (Fibly et al, 2010). For example, Larson, OMalley, and Melloni Jr from Northeastern University determined a dominant and subordinate relationship between zebrafish by measuring aggressive attacks after fish have been separated for five days (Larson et al, 2006). By the fifth day of observation, the dominant-subordinate relationship was firmly established (Larson et al, 2006). The dominant male patrols the tank looking for food, while the subordinate remains relatively stationary in the corner of the tank (Larson et al, 2006). Similarly, Huntingford et al. found that the larger the size of Atlantic salmon directly correlates to social dominance, and those larger fish reaped benefited from high status, such as more access to food (Huntingford et al, 1989).
The Nano community being studying is held in an aquarium replicating their natural environment. In home aquaria surrounded by other aquaculture species, Neon Tetra tends to display reduced aggression and darting, and spends more time shoaling in larger groups because they become more familiar with their environment and fear is reduced (Saxby et al, 2010). Cichlids have been found to be rather calm and reclusive (Hert 1995). When Cichlids were transported from Lake Malawi to artificial miniature reefs with established species, they were found to coexist well in a site with a diversity of species (Hert 1995). Furthermore, a heterospecifc neighbor is less likely to interfere in courting and spawning which leads to male-male competition avoidance (Hert 1995). The variations in boldness and aggression between the same species of vertebrae fish have been linked to variation between genes (Norton et al, 2011). The discrepancy between the fish of this nano community is not only dependent on physical attributes like size but genetically dependent. It is hypothesized that a social hierarchy will exist. The largest species of fish, the Hatchet, is expected to be the most aggressive and therefore the highest standing on the social hierarchy. In contrast, the smallest species, dawn tetra, will be more submissive, resulting in a low standing on the social hierarchy.
Rank in a hierarchy can be influenced by a wide variety of qualities: behavior, physiology, health, and the ability to produce offspring (Chase et al, 2002). Aggression is a primary behavior that correlates with dominance in a group. A combination of hormones, neurons, and genes can be used to explain aggressiveness in organisms (Jones et al, 2014). The utilization of genetics is efficient for further investigation of the homologous behaviors that exist between the fish species of the Nano community and humans. Zebrafish are a part of the class actinopterygii; the same class as the fish species in the nano community (Norton et al, 2011). Zebrafish are being utilized more frequently in medical research because of the 60% genetic identity they share with the human genome (Arslan et al, 2010). Monoamine oxidase, a gene linked with aggression, is found in both humans and actinopterygii species (Anichtchik et al, 2006). The MAO-A gene is found on the X chromosome at location 11.3 and has 16 exons. The MAO gene is involved with oxidative deamination of amines such as serotonin, norepinephrine, and dopamine. The breakdown of these amines has the capability to influence feelings, mood, and behavior. (Arslan et al, 2010). A single MAO gene in zebrafish, zMOA, shares approximately 70% sequence identity with human MAO-A. (Anichtchik et al, 2006). In both humans and zebrafish, if there are insufficient levels of MAO, the levels of neurotransmitters in the body can fluctuate. Therefore, the functionality of the MAO gene is what regulates behavior in these organisms. (Arslan et al, 2010).
Methods
Finalized by: Lindsey Hood
Focal Sampling
Four species of small fish: Blue Ram Cichlid, Hatchet, Dawn Tetra, Lamb Chop Rasbora occupying a nano community tank in the Lyman Briggs biology laboratory were observed. Although several fish of each species resided in the tank, only the largest fish of each species was used for observation. Focal sampling will be utilized meaning the largest fish will be used to determine dominant behaviors because larger fish have shown to be more aggressive and dominant. Nolan found that the two most dominant fish species in his tank of study were the two largest in size. Agonistic behavior was selected because establishing dominance should involve some form of aggressive action towards other fish (Nolan, 2010). Then with varying sizes between species, the dominant hierarchy seeks to determine whether there is a correlation between size and dominance is a nano community. Determining the largest fish was done qualitatively with the notion that size can influence one’s ability to exhibit dominant behavior. Comparing a particular fish’s size to the other fish of the same species allowed for the determination of the largest of each group. To ensure that the same four fish were observed each time, unique physical attributes along with photographs taken on a cellular device were used for future reference. For example, the largest Lambchop Rasbora had a significantly larger stomach with a distinct black stripe going from underneath the stomach to the tail. This was noted in a notebook in addition to a picture taken on a cell phone. Before each observation, these were referenced to ensure that the same fish was observed each time.
Time Interval
Each observation period consisted of 15 minutes, occurring twice a week. The 15 minute time interval was selected because a similar study describes an undergrad biology class that observed a communal fish tank for aggressive behaviors. Each student in the class observed one of four focal fish for 30 minutes because this was half of their lab time and proved to be plenty of time to gather data (Nolan, 2010). If the observation period was too short, it is possible that no aggressive behavior could be expressed and therefore not produce results which is why a shorter period was not chosen. It was decided that observation period for the nano community fish tank would last 15 minutes instead of 30 due to a reduced amount of people able to observe and due the fact that Nolan found 30 minutes to more than enough time to collect data. For 15 minutes, each group member observed the focal fish of one species. Observations were done in the Lyman Briggs biology lab in regards to the nano community tank. The fish tank was kept in its natural state, with no manipulation of the original environment.
Behavioral Sampling and Data Recording
This study was purely observational; therefore, focal sampling and behavior sampling techniques were utilized throughout the 10 weeks of observing. Behavioral sampling simply means only recording data when a specific behavior occurred. The behaviors that were observed were: chase, spar, repel, and attack. These methods avoid any type of manipulation of the animals and provide a more specified route of observation. Chase was any fish that swam directly towards another fish, following them with increased speed. If a behavior was described as spar, this meant the fish slowly approached another fish head on with its fins flared to give the illusion of a larger size. Behavior was classified as repel if a fish made any sudden movement that caused approaching fish to change their swimming direction. Finally, attack was any time a fish bit or nipped at another fish. If the focal fish was the one performing these behaviors - chasing, attacking, repelling, sparing - this was considered a dominant behavior. However, if the fish was the one being chased, attacked, etc. then this was considered a subordinate behavior. While observing the focal fish, the number of times it exhibited any of the four behaviors was recorded with a tally system in a notebook. The tally system is defined as: a chart with the title being the species that is observed at the time, then the four behaviors listed as rows, and “dominant” or “subordinate” columns at the top. Each time the focal fish performed an aggressive behavior there was a tally mark listed under that behavior in the dominant column. If the fish exhibited a subordinate behavior a tally mark was listed under that behavior in the subordinate column (Nolan, 2010). At the end of each 15-minute period, the observed fish was either classified as “dominant” or “subordinate”, depending on whether the fish had more dominant or subordinate behaviors. At the end of the 10 week period, all the dominant and subordinate behaviors were added up for each focal fish. Using statistical analysis, a chi square test was preformed to compare results of dominant vs subordinate attacks to analyze the distribution of a data set versus the assumption there will be equal distribution among the possibilities. In other words, if there is no hierarchical organization, then the attacker and defender events should be occurring at equal frequencies across all fish (Nolan, 2010) (see Statistical Analysis). Using a cell phone camera, videos were taken of the fish as a way to document behavior. Along with videos, a notebook was used for additional observations regarding the focal fish, such as where the fish was mainly swimming for that day. This was repeated for each of the four focal fish.
Homologous Behavior in Humans
To further investigate social hierarchies in different species, human behavior in a social setting was observed. This data collection seeks to determine whether humans form a social hierarchy between the members of their party while dining, and whether dominance corresponded to size; For example, whether a large male is more dominant than his smaller son. Human subjects were observed in various restaurants in East Lansing such as Harrison Road House, Buffalo Wild Wings, and Hop Cat, paying close attention to the behavior of the subjects before ordering, while eating, and after eating. The four observed behaviors were: who decides when they are ready to order, who orders the appetizers or any other food for another member, who flags the waiter down if they are in need of anything, and who pays for the bill. Again the same tally system was used whenever a behavior was exhibited by a member of the party (see Behavior Sampling and Data Collection): a chart was created for each member of the party, the title of the chart was a description of the relative size, age, and gender of the human, the rows on the left of the chart were the four observed behaviors, and across the top were dominant and subordinate columns.
DNA Comparison
Understanding the genetic contribution to aggressive behaviors in both species was explored through DNA testing. Human DNA was obtained from the biology department at Michigan State University. Similarly, fish DNA was obtained from a scale supplied by researchers and tested for comparison with human DNA. Polymerase Chain Reaction testing was used to make copies of particular sections of the DNA. PCR testing requires the DNA template to be copied, primers, DNA nucleotide bases, Taq polymerase enzyme, and a buffer. PCR utilizes techniques such as denaturing, annealing, and extending. During the denaturing process, DNA double-strand template is heated to separate the DNA into two single strands. The temperature is lowered during annealing, which enables the DNA primers to attach to the template DNA. The temperature is raised again during extending when the new strand of DNA is made by the Taq polymerase enzyme. After this process, the mixtures are electrophoresed with gel in order to produce a genotype frequency. To test for the MAO gene, cDNA was obtained and amplified. Using custom primers, PCR was run. The forward primer 5’-CCGGATCCATGACTGCGAACGCATACGAC-3’ and reverse primer 5’-GGGCAATTCTTAACACCGTGGTGGAGGAGCCC-3’ were used. The PCR conditions were the following: 95 degrees Celsius for 1 minute, and then 35 cycles at 95 degrees Celsius for 50 seconds, 60 degrees Celsius for 50 seconds, and 72 degrees Celsius for 2 minutes. Afterwards, Taq DNA polymerase was added at 72 degrees Celsius with a final extension time of 15 minutes. The MAO gene was amplified and identified on agarose gel electrophoresis. A DNA sequence analysis was performed to confirm the orientation and sequence of the gene. (Arslan et al, 2010).
Statistical Analysis
The collected data was subject to statistical analysis using the Chi-squared (x2) test. The difference between the dominant and subordinate behaviors was compared to the null hypothesis which is the assumption there will be equal distribution among all the possibilities. Meaning, if there is no hierarchical organization, then the attacker and defender events should be occurring at equal frequencies across all fish (Nolan, 2010). The x2 test was performed by adding the total dominant behavior for each fish in one column, and separately adding all subordinate behavior for each fish in another column, resulting in the observed data. The expected data was calculated for both dominant and subordinate behaviors for each fish by multiplying the observed data by the total behaviors for the fish, and dividing by the total of the observed data column (either dominant of subordinate behaviors). The x2 results were calculate by subtracting the expected results from the observed results, squaring, and dividing by the expected data. The x2 test with 7 degrees of freedom revealed that the dominant behaviors of the hatchet x2 = 19.39 (p < 0.05) were statistically significant, as well as the subordinate behaviors of the hatchet x2 = 33.88 (p < 0.05), and the subordinate data for the lamb chop rasbora x2 = 22.45 (p < 0.05). The null hypothesis was therefore rejected. There is not equal distribution between all species of fish. The data from this test supports the idea that there is a complex social structure present in the tank.
Results
Finalized by: Olivia Bayer
The depictions of the four different species of fish show the relative size of the hatchet, lamb chop rasbora, dawn tetra and blue ram cichlid (Figure 1). These fish were observed for 15 minutes in the Lyman Briggs biology lab. Dominant behaviors are observed when a fish is performing a chase, repel, spar or attack on another. On the other hand, a subordinate behavior is having one of these four behaviors executed on the focal fish. This study found that the larger fish, such as the Hatchet had the most dominant attacks, 114 by the end of 10 weeks, and smaller fish such as the lamb chop rasbora had the most frequent subordinate behaviors, 67 by the end of 10 weeks compared to the Hatchets 5 subordinate behaviors. There was not equal distribution between the fish, resulting in a correlation between size of the fish and the number of observed aggressive behaviors (Figure 1).
After 10 weeks of studying the Hatchet, Lambchop Rasbora, Dawn Tetra, and Blue Ram Cichlid the dominant behavioral data was collected after every week (Figure 2). Aggressive behaviors - such as chase, spar, repel and attack – were added up after each week for each of the focal fish. The previous week was independent of the following weeks. Here, the data shows a lack of randomness between aggressive behaviors, and instead a steady hierarchy persisted week after week. The Hatchet is consistently the most dominant species, surpassing all other species by at least 4 aggressive acts except for week 6. Until week 7, the blue ram cichlid was continuously failing to interact with the other fish. As time went on the cichlid performed more aggressive acts and in week 10 the cichlid was the second most aggressive fish. The Lambchop Rasbora executed the second most total aggressive behaviors in 6 of the 10 weeks. Unlike the Cichlid, the Rasbora was very interactive with other fish. Not only did this fish have the second most aggressive acts, but the most subordinate behaviors (Figure 2).
The data collected demonstrates an uneven distribution of the number of aggressive versus subordinate behaviors (Figure 3). The largest fish, the Hatchet, stands out because this fish had the most aggressive behaviors, and was attacked the least often out of all the other fish, except the Blue Ram Cichlid, which was only the subordinate once. The dominant to subordinate ratio was greatest for the Hatchet (114:5 or 22.8). The second largest fish was the Blue Ram Cichlid which also had the second largest dominant/subordinate ratio (11:1 or 11). The Cichlid was the largest in size but had the least number of overserved behaviors out of all the fish. It was not until the end of the study that the cichlid became more active. The Lambchop Rasbora (37:67 or .55) and the Dawn Tetra (25:43 or .58), the third and fourth largest fish respectively, received more attacks as the subordinate than dominant behaviors which resulted in a ratio less than one. The Lambchop Rasbora had nearly the same amount of observed behaviors as the hatchet yet a larger portion of the Rasbora;s behaviors were subordinate (Figure 3)
The difference score is the compilation of dominant behaviors performed by each focal fish compared to the subordinate behaviors performed by the same fish (Figure 4). In other words, Figure 4 depicts the number of times the fish was the attacker versus the number of times that each fish was attacked by any fish in the tank. A positive difference score corresponds to a dominant fish and a negative difference score corresponds to a subordinate fish (Nolan, 2010). The zero line on the y-axis depicts that either the fish had no interaction with other fish, or there were equal amounts of dominant and subordinate behaviors. This graph most accurately depicts that a hierarchy that exists between the fish. Yet, this existing hierarchy is not assumed to be linear because it is possible to see what appears to be near-linear that is due to change factors which would be more solidified with more tests (Nolan, 2010). Statistical data, using a x2 test, rejected the null hypothesis. The testing for dominant behaviors in hatchet fish x2 = 22.45 (p < 0.05), the subordinate behaviors in hatchet fish x2 = 33.88 (p < 0.05), and the subordinate Lambchop Rasbora x2 = 22.45 (p < 0.05) all proved to be statistically significant (Figure 4).
References
Finalized by: Olivia Bayer
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Figures
Finalized by: Lindsey Hood
Figure 1: Images of the Four Species in Nano Community. The four species of fish in the nano community (pictured left to right starting in the top left corner): hatchet, lamb chop rasbora, dawn tetra, and blue ram cichlid. All four are freshwater fish and are depicted in aquaria. In order to determine a social hierarchy, focal and behavior sampling will be unitized, meaning the largest fish in each species will be observed for aggressive behaviors. Pictures and videos are taken every observation period and are crucial to ensure the same focal fish are observed. Here the sizes of the fish can be seen, with the hatchet fish being the largest and the tetra being the smallest. The larger species, the hatchet fish, exhibited more dominant behaviors than any other species.
Figure 2: Dominant Behaviors Observed in the Nano Community. Over the course of 10 weeks dominant behaviors were observed in the nano community fish tank. The four behaviors observed were chase, repel, spar and attack. The fish was either the aggressor or subordinate in each of the listed behaviors, depending on whether the fish was the one performing these behaviors or having these behaviors performed on them. All four aggressive behaviors were counted after each week for each of the four fish to determine which species executed the most dominant behaviors. The previous week’s aggressive behaviors were not counted in the following weeks.
Figure 3: Dominant vs Subordinate Behaviors. The bar graph indicates the total number of times that each fish was the dominant – executing aggressive behaviors towards other fish – or the subordinate – the fish that the aggressor attacked. The dominant and subordinate behaviors were documented for 15 minutes in a nano community observing for the behaviors: chase, repel, spar and attack. This data was used to perform a chi-squared (x2) test to determine if there was significance in the occurrence of these aggressive or subordinate behaviors. The x2 results prove that the dominant and subordinate behaviors of the hatchet fish, as well as the subordinate behavior of the rasbora were statistically significant (p < 0.05). This rejected the null hypothesis that all behaviors have equal and random occurrences.
Figure 4: The Difference Score. Graph indicates the number of times the fish was the dominant aggressor versus the number of times that each fish was attacked by any fish in the tank. The number of aggressive behaviors observed: chase, spar, repel, and attack were totaled after 10 weeks for each fish. Similarly, the amount of subordinate behaviors were totaled after 10 weeks. The subordinate behaviors were subtracted from the dominant behaviors. The zero line on the y-axis implies neither aggressive nor subordinate behaviors were performed or that the fish performed equal amounts of aggressive and subordinate behaviors. A positive level of dominance corresponds to a fish with more dominant behaviors than subordinate, and a negative level of dominance corresponds to a fish with more subordinate behaviors (Nolan, 2010). This graph most clearly depicts the hierarchy that exists within the nano community.
Documentary Video: This documentary outlines our journey while studying the fish species in our Nano Community.