Observing the Fright Response of Etheostoma Blennioides When Exposed to Unfamiliar Sounds.
By Mike Martinson, Joe Oehrli, and Tanner Porter
LB 144 Cell and Organismal Biology
Thursday 7PM
Morgan Kiryakoza and Mellissa Ungkuldee
December 1, 2016
https://msu.edu/~mart1541/
https://www.youtube.com/embed/XH47egjhpwo
(Title page written by: Joe Oehrli
Natural sound in the environment is crucial to the survival of the organisms that live in it since sound is a form of universal communication between the different organisms in the environment (Voellmy, Irene K., et al, 2014). Changing or removing theses basic communication elements from an environment can cause stress to the organisms since sound under water travels farther faster and has less attenuation (Nichols, et al., 2015). It is due to these changes in the behavior of sound underwater that aquatic organisms are much more affected by changes to sound in their environment.
Stress levels in fish are measured by the amount of the hormone Cortisol inside the blood stream (Nichols, et al., 2015). Cortisol is released by the adrenal cortex of the fish’s brain in times of extreme stress, and allows for the conversion of lipids into energy for faster movements to avoid predation and dangerous situations (Nichols, et al., 2015). This consumption of lipids is observable as fast and erratic movement and changes in a fish’s behavior (Nichols, et al., 2015). This change in behavior is key when looking at the green-sided darter, Etheostoma blennioides. This demersal freshwater fish remains relatively still on the bottom of rivers only moving when it feels threatened. It is when the darter perceives a threat that cortisol is released into the bloodstream and used to produce energy from lipids to avoid predation. Human reactions to these ambient noises had produced multiple reactions, a few worth noting were stopping to see what the noise was, puzzled looks or gestures and verbal acknowledgement of an alien noise.
Using information obtained from research by Nichols, et al., 2015, who had done research on boat motor frequencies and their effects on giant kelp fish by placing the kelp fish into fish tanks where they then played three different boat motor frequencies, intermittent, static and continuous frequency. They then humanly killed the fish by clubbing the fish and taking samples of their blood to measure the levels of cortisol. They had found that the intermittent frequency had the highest level of cortisol produced, therefore coming to the conclusion that the intermittent frequency had caused the most stress to the fish. We had hypothesized that the cortisol levels of the Etheostoma blennioides will increase the most during the intermittent sound because of the unpredictable noise patterns. We had decided to perform the experiment in a similar way to Nicholas, et al. but with a few changes to the methods, instead of clubbing the fish to measure cortisol levels we instead observed the darters movements in reaction to the boat sounds and documented the movements.
CYP17A1 is the gene that controls and regulates cortisol production in humans is located on chromosome ten (Kniffin, 2005). CYP17A1 codes for the enzyme CYP17A1 which regulates the production of cholesterol into steroids such as cortisol (U.S. National Library of Medicine, 2016). If these hormones provide the same effect in both humans and green-sided darters, then it is possible that humans and green-sided darters share the CYP17A1 gene.
Six Etheostoma blennioides were captured via nets from the Red Cedar River in East Lansing, Michigan on September 19th 2016. The aquarium was held at a temperature ranging for 65-75 degrees Celsius, the water was regularly filtered from a built in Revco filtration system. The fish fed on bloodworms once a day. They were in contact with a single rock bass (Ambloplites rupestris) also found in the red cedar. Ambloplites rupestris when fully grown is a natural predator of the green-sided darter and this predatory behavior was expressed during the observation periods in the form of chasing darters. The environment of the aquarium contained green colored pebbles, a piece of wood, three medium sized rocks, and four plastic grasses like structures to resemble that of the red cedar as closely as possible. The fish showed signs of adequate health and the sex of the fish was determined to be equal numbers of each sex. The source for boat engine noises was from a recording of a small fishing boat located on a small river similar to the red Cedar via youtube. Boat noises were recorded one meter away from the boat's engine, the total amount of time recorded was forty seconds (Edic, 2014). The pattern for the boat noise was static at first then starting the engine and having it run smoothly for twenty seconds then accelerating and decelerating the throttle making a variety of noises (Edic, 2014). The recordings were then split into three categories, static, constant throttle, and varying throttle. The recordings were also set on a loop to make them last longer. Then once the recordings were separated they were analyzed for their frequency level by turning the recordings into mp3 files and uploading them to an online program Audacity which shows the frequency levels of the mp3 file (Voellmy etal.,. 2014).
The setup of the experiment was done by suspending a PowerLead BP013 IP67 underwater speaker upside down by a suction cup directly in the center of the tank to ensure no added vibrations from contact from the fish tank and to minimize any possible resistance (Voellmy et al, 2014). We then placed the speaker under the water so it would protrude three centimeters into the water from the middle of the tank we then wirelessly connected an iPhone 6 to the speakers via Bluetooth and played each of the three sounds twice at max volume using the voice recording on IOS 10. Each of the trials lasted five minutes and breaks in between trials lasted until the darters resumed normal laxative behavior. While sitting three meters from the tank we recorded their movements either irritated or stressful movements (either a side to side or a vertical movement) for five minutes. Repeating this process twice both each on a separate day at the same time of day to ensure that the fish would continue their normal living habits. The control to the experiment was the static recording. The reason why we used this as the control was to ensure that the speaker alone was not causing any added stress to the fish (Nichols et al, 2015).
After the observation periods are concluded the reactions of the darters is recorded in the lab notebook and the movement and behavior of the darter is used to determine how much of an affect the sounds had on the individual fish. Video recordings of the experiment were taken using and iPhone to ensure that all procedures were followed and that the reactions of the darters was captured for further interpretation. When looking at the correlation between how organisms interpret and respond to different sounds in the environment it is logical to look at humans as well. In this experiment individuals were given a set of headphones which played a variety of familiar and unfamiliar sounds for five minutes. During this time the reactions of the individuals was recorded in the lab notebook which could later be used by the researchers to determine the magnitude and affect that each of the sound had on the individuals.
When looking at the human studies an observational experiment was conducted in which individuals where exposed to the same five minute sounds that the darters were exposed to. The response of the individuals was recorded and sorted into three different categories: neutral, positive, and negative. The experiment was conducted at three different locations and the PowerLead BP013 IP67 speaker was hidden within ten feet of the observers while remaining out of immediately out of sight. We then will look into the PEPCK gene located in the fish and see if the sequence use in Mathilakath et al. paper and see if we could find a similar sequence in the human genome using the NCIB website used in lecture(Mathilakath et al, 2003).
(Figure 1) shows the lack of horizontal and vertical movements when there is a lack of boat engine noise being played. Based on the experiments performed by Nicholas and Voellmy, there seems to be a very specific pattern. The pattern is, stress levels go up in fish as you randomize and increase the frequency being played (Nichols et al, 2015). We predict that there will be the most increased movements both lateral and horizontal of the greensided darter in figure 1 for the intermittent category because it is the most randomized and highest frequency sound being played(Nichols et al, 2015). This will happen because the darters will be able to get accustomed to the pattern of the static and constant playback sound, but with the randomized intermittent category, the darters will not be able to get used to the pattern because there is none (Nichols et al, 2015).
(Figure 2) shows the increase in which the darters stress occurs when introduced to these sounds. When looking at the two experiments by Nicholas and Voellmy, both showed that stress levels in fish go up when alien events occur. According to Nichols and his companions, the kelpfish that they studied with boat frequencies in the shipping channel the pulsating frequency caused the most stress (Nichols et al, 2015). We predict that the pulsating frequency will create the highest amount of stress in the green sided darter because based on Nicholas’s similar study showing the highest added stress to the kelpfish (Nichols et al, 2015). Figure 2 shows the added stress (in percentage) added to each of the three darters.
In (Figure 3) we look at percent stress over time. Based on the experiments preformed my Nicholas and Voellmy there is a trend to be seen in the responses of the fish studied to the sounds of the boat motor. As the sounds became more random and less predictable the stress and amount of cortisol in the fish’s blood stream increased compared to the sound of the motor being run consistently. We predict that this trend will continue in the green-sided darters because of the randomness and unfamiliarity resulting in higher levels of cortisol in the bloodstream (Nichols et al, 2015). In our study our data clearly pointed to the same results. The darters had far more movements when exposed to the random interval noise when compared to static and constant noise.
(Tanner Porter) Figure 1- Lack of movement of green sided darters in absence of stimuli. Here we are looking at a thirty second video showing the behavior of the green sided darters being used in the playback experiment. They show a specific behavior of hovering over the very bottom of the tank and move occasionally but not frequently whatsoever. This is because when there is a lack of stimuli the fish will go into a relaxed less active state where it can save and preserve energy for when a predator might come up, so it can flee and survive. This lack of movements when there is no stimuli is an important control for our experiment for when we have the playback noises going we can compare it to the the amount of movement that the fish had when there was no stimuli(Nicholas et al, 2015).
(Joe Oehrli) Figure 2- Looking at the Stress Influx by Different Motor Sounds on Fish, off of the project by Nicholas et, al. 2015 noting that fish seemed to be less stressed by the continuous motor sound. The continous motor sound is composed of a ten minute long audio recording of a consistantly running boat motor underwater.The fish appeared more stressed by the pulsating sound. Which is composed of a ten minute audio recording presenting random interpitant sounds of a boat motor stopping and starting. We predict that the green sided darter’s will be more stressed by the pulsating sound. By suspending a water proof, bluetooth speaker into the water, after letting the darters get used to the control which is the static sound, we will then wirelessly play the different sounds. We will then observe their changes in movement, noting horizontal and vertical movements of the green sided darters as well as their behavior for five minutes and record the data. Once all of the observed data has been recorded we will then preform a Chi squared statistical analysis.
(Mike Martinson) Figure 3- Predicting the results of a playback experiment in which green-sided darters are exposed to different sounds of boat motor sounds for a ten-minute period. Over this time the darters will be observed and their swimming patterns will be analyzed to produce a baseline for statistical tests. We predict that as the sounds of the boat motor become louder and more random over the ten-minute period that the darter’s level of stress will increase due to the amount of cortisol in their blood stream (Nichols et al, 2015). Figure Video
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