Observations of Fish Interspecific Interactions as the Cause of Color Changing and Behavioral Stress Responses in the Blue Ram Cichlid





























By: Jacy Wacker, Brandon Blasius, Mitch Konieczny, and Claire Boudreau




























LB 144 Cell & Organismal Biology
Thursday 7 PM
Roshan Angoshtari, Morgan Kiryakoza, Mellissa Ungkuldee
12/1/2016
https://www.youtube.com/watch?v=Ws3jLwzaLEk&t=1s
Team E=MSU
(Title page written by: A52263942)

Introduction


Written By: A52263942

   The fish species studied in this experiment is the Mikrogeophagus ramirez. The common name for it is the Blue Ram Cichlid. They live 3 years or more. The coloration of this species is a vibrant coloration. It is a stocky fish with bright grey and blue flanks. The head and chest of this species of fish is yellow, gold, and black with blue. The belly is red in color. This species also has a black line running across the eye and has a red patch around it. The dorsal fin is yellow in color, while the pelvic fins are red and blue. They grow to be between two and three inches. The Blue Ram Cichlid originate from the rivers of Venezuela and Columbia. The temperatures need for this fish to survive are seventy eight and eighty one degrees farenheit. The pH levels that they need to survive are 5.5-7.0. They are also omnivores. They prefer to eat frozen bloodworms and brine shrimp and other live foods. The females in this species are bigger than they males and their tails are more round edge on it. The red patch on their stomachs is also bigger than males. During breeding the red patch gets brighter. The male dorsal and anal fins have a more pointed edge. Their tails also have a V shape. For the eggs their pH level needs to be between 5.5 and 6.5. The Blue Ram Cichlid reaches maturity between 4 and 6 months old. Females lay between 20-200 eggs. Both parents will attend to the eggs. Sometimes they will also eat their eggs especially the infertile ones (FishLore, n.d.). Even though they sometimes eat their own eggs, the Blue Ram Chiclid is not an aggressive species.



  The reason we can expect signs of aggression to be infrequent is because the Blue Ram Cichlid is a relatively docile member of the cichlid family. Fish occupying an overlapping territory compete for the same resources necessary for reproductive success including limited space, food, and shelter. When environmental variation is low, the competition for these resources becomes more intense (Grossman et al., 1998). Aquarium limitations further constrain occupation and reduce resource availability. The competition for resources creates an intra- and interspecific dominance hierarchy and results in interactions between occupants of the space (Connell, 1978). Low social status among fish has a great impact. In rainbow trout, social stress suppresses the immune system, in cichlid fish growth is slowed, and in some marine environments social stress causes female fish to have smaller offspring (Peters et al, 1991; Hofmann et al., 1999; McCormick, 2006). Behavioral indicators of social stress can sometimes be hard to observe. Acute social stress, such as aggressive interactions, dont just cause behavioral changes, but can cause physiological responses as well (Clement et al., 2005).



   We predict that some behaviors we will observe for the Blue Ram Cichlid's response to stress from the environment/other fish will most likely mirror that of similar fish species. There are many species of fish that change color when introduced to stress (usually from a brighter, original color to a paler shade of the original) (Braastad et al., 2009). Some fish species will quickly change their depth in the water to avoid a particular stress, and some fish get dark colored circles around their eyes when stressed, known as eye darkening (Freitas et al., 2014). We also plan to perform oberservations on people to see how they exhibit stress-related behaviors in stressful environments, and see how they correlate to those exhibited by the fish. The stressful environments we intend to observe humans in are what we believe to be universally stressful to a majority of people, such as public speaking, or dealing with crowds. The behaviors we plan on observing in humans are people removing themselves from stressful situations to limit or stop the amount of incoming stress, and changes in color due to an increased amount of stress, (e.g. increased/decreased reddening of the skin) (Gertrude and Hearing, 2007).



   Physiological changes, such as reddening of the skin, are the most easily recognizable indicators of stress in blue ram cichlids. Eye darkening is one of these reliable and easy indicator of stress within fish. Researchers have found a connection between eye darkening in some fish and confinement and we believe this same connection exists in the Blue Ram Cichlid (Frietas et al., 2014). By checking the eye darkening we can easily determine if the fish is stressed and by how much. This is determined by the size of the darkening within the eyes. Another indication is by the coloration of the species. Different levels of stress can cause the coloration to fade or become glossy (Rose et al.,2008). A severe lack of color is a great and reliable indication that the specimen is under heavy amounts of stress. By observing them, one can determine where the stress is coming from. We hypothesize that if the Blue Ram Cichlid is faced with interspecific stress interactions then a physical color or behavioral change will occur due to an increase in cortisol production. A major and well known stress indicator is gill cortisol concentrations. Cortisol is the main biomarker of stress in fish (Gesto et al., 2015). Some non-invasive techniques have been developed to measure cortisol directly in the water from fish-holding tanks, in skin mucus or in scales (Koakoski et al., 2012). Commonly measured reactions include change in levels of plasma corticosteroids and noradrenaline (Bonga, 1997; Gilmour, 2005). Of the research experiments we have cited, there are some we found that point to a genetic component in cortisol responsiveness.



   The increase of cortisol due to stress is regulated by the hormone corticoid-releasing factor (Fevolden et al., 1993). Many studies show that this cortisol level response as a reaction to stress is genetic. In rainbow trout, there is a heritable difference in brain chemistry between trout who have high-responsiveness and low-responsiveness to cortisol levels (Orveli et al., 2006). Atlantic salmon show the same heritability of cortisol stress responses (Fevolden et al., 1990). In sea bass, the stress response genes has been determined and primers have been created (Gornati et al., 2004). In humans, exposure to acute stressors, although different stressors than fish are exposed to, is correlated with higher levels of cortisol (Dickerson and Kemeny, 2004). The serotonin transporter polymorphism (5-HTTLPR) in combination with the environment, is shown to increase cortisol levels in reaction to acute stress in humans (Alexander et al., 2009). This manifests as a higher likelihood of the development of depression or anxiety traits (Sen et al., 2004).

Methods


Written By: A53180551
Equipment


   Before any tests could be done, a video recorder, camera, and fish were needed. The two Blue Ram Cichlids (Mikrogeophagus ramirezi) were in a 25+ gallon tank with 10+ total fish. We also used a camera to have a capture and record the movement and interactions between the Blue Ram Cichlid, Dawn Tetra (Aphyocharax paraguayensis), Lambchop Rasbora (Trigonostigma espei), and the Hatchetfish (Sternoptychinae) so that we could review it while we are away from the actual fish. The video recorder was on the entire 15 minutes of observation to capture all exhibited behaviors of the blue ram cichlid. Artificial lighting from a lightbulb was also provided to simulate natural lighting and heating. Within the tank there was a common bubble stone to provide oxygen, and a filter which is essential for the species to live.

Observations

  We are observing the two blue ram cichlids that are contained in the Nano Community. One of the blue ram cichlids is a male while the other is a female. We will be observing the stress behaviors exhibited by the blue ram cichlids as they interact not only with each other but with the other fish in the tank. The Nano Community is an aquarium community that consists of the Blue Ram Cichlids, Hatchet Fish, Dawn Tetra, and the Lamb Chop Rasbora. The group will be observing them two days of the week. Those days will be Tuesday and Thursday. The observation will take place at the same time of day; around 6:45 pm. The observations will take place for fifteen minutes. The group will be looking for any stress indicator behavior exhibited by either of the Blue Ram Cichlids, then the group will record any of those stress indicators. Observations will be recorded on camera through the live stream or on a cell phone. Observations will also be recorded on paper.

Measurements


  While stress can be measured empirically by taking whole body samples to measure cortisol levels, we opted to pursue a less invasive, observational study. Our focus on the acute interactions rather than long-term social hierarchies led us to reject the documentation of growth and reproductive indicators that also demonstrate stress. Of the many tertiary responses to acute stress, we have selected to emphasize on color changes in the blue ram cichlid's skin and eye-darkening; selected for the ease of measurement and the ability to easily distinguish the reactions from normal behaviors. We will use a method for eye-darkening similar to that used when measuring eye-darkening in Nile tilapia, that divides the eye into eight equal sections, each 12.5% of the total eye, and uses this to estimate the total percentage of eye-darkening (Volpato et al., 2003). We will be monitoring temperature and lighting to ensure that they remain constant and do not contribute to a variation in the stress of the blue ram cichlids. We will also observe fish who are not interacting for equal interval as a baseline measure.

Data Analysis


   Data for the stress-related behavior was analyzed by using the chi-squared test to determine a percentage relation between the observed number of occurences of each behavior, and the expected number of occurrences of that behavior. As Figure 3 shows, we observed the number of times the Blue Ram chased the Dawn Tetra to be 2 times, and we also observed the Blue Ram flee from the Dawn Tetra 4 times, and shoaled with it zero times. In regards to the female Blue Ram, the male chased it 5 times, fleed from it zero times, and shoaled with it 8 times. When calculated, the chi-square value is about 12.43, and this gives us a p-value of less than .01, meaing that the difference between the expressed behaviors is statistically significant. For the skin color of the Blue Ram Cichlid, to analyze its level of stress we used a software known as ANOVA test.

Results


Written By: A48778306

   In the figure 1, it shows clear evidence that the male and female Blue Ram Cichlids change different colors based on their gender. The female Blue Ram Cichlid changes from a blue to a pale blue. The female was in a blue when she was put under a non-stressful situation. But after the male was put into the tank she turned a very pale, translucent color (Figure 1). Now the male Blue Ram Cichlid was a blue color also before he was put into a stressful situation. After he was put into stressful situation, his body color turned a brighter blue (Figure 1). A statistical test was run on the amount of times that we saw a color change between the two genders. The test run was a t-test. The results came out to .64, which in turn makes the data collected insignificant but since this was based off of color changes based on pictures. We set the test results aside and focused on the color changes based off of the pictures.



   The capacity for physiological color change in Mikrogeophagus ramirezi was extensive (Figure 2). Across all sets of data of the Blue Ram Cichlid color brightness ranged from 207-36 with 36 being the darkest blue, and 207 being the brightest. The colors range from dark blue with a brightness of 36, medium blue with a brightness of 53, true blue with a brightness of 66, sky blue with a brightness of 193, and light blue with a brightness of 207. To determine the color of each picture, which was our data, an HTML color coding was used (“Get Colors from Image”). After the color was determined, a brightness chart was referenced to determine a number of brightness per data set (“Color Hue”), A one-way ANOVA test was used to determine the mean brightness of 120 which correlates to the color Dark Cyan (Rose et al., 2008). The standard deviation of the date is 69.69 with a standard error of 13.94. Changes in brightness were correlated with being chased by another species within the aquarium. When very little to no chasing occurred, the Blue Ram Cichlids color ranged from brightness of 53 -193. When chasing did occur two brightnesses were shown. The dark blue with a brightness of 36, and the light blue with a brightness 207.



   Our initial prediction was that the Blue Ram Cichlids will exhibit behaviors that are clear indicators of stress from the presence and atitude of the other fish inside the aquarium. Our reserach found that, while relatively docile and inactive, the Blue Ram Cichlid did exhibit a variety of behaviors towards the other species, such as the Dawn Tetra, in the aquarium. Species of fish will act differently according to how many fish are around them, and how those fish perceive each other (Clement et al., 2005). When there is stress between fish they will exhibit certain behaviors that act as indicators of their raised stress levels; these behaviors include fleeing, shoaling, and chasing. We initially predicted that the Blue Ram Cichlid would exhibit a mixture of these behaviors towards the Dawn Tetrta, but that it would not demonstrate an increased level of tension towards the other Blue Ram Cichlid, which is of the opposite sex, (female). Our results, as shown in Figure 3, show this prediction to be correct (Figure 3).



   In the human observation trials we were able to observe multiple signs of stress while giving a speech. These signs include redness in the face, sporadic hand movements, and fluctuating frequencies in voice (Sen et al., 2004). These behaviors were analyzed and ranked by importance on how much they expressed the amount of stress. The rank of how much each behavioral sign starts with different frequencies in voice having the most effect on stress and redness in the face having the least amount of effect (Mathews 1978). By using a t test we were able to calculate a mean of 7.49 by using a scale from 1-3 of importance. With these observations we were able to conclude that a small amount of stress and anxiety were both observed in our experiment (Mathews 1978).

Figures


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Figure 4: Blue Ram Cichlid Documentary

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Appendix