The Effects of Increased Salinity, Temperature, and Ocean Acidity on the Behavior and Health of the Common Clownfish (Amphiprion ocellaris)

Clownfish and Climate Change:

By April Dawson

High School Senior

Boulder High School Boulder, Colorado

Research has only recently been conducted on fish and the effect that ocean acidification has on them. Clownfish and damsels are often used as representative species because they are easy to keep in captivity. As was described in my introductory video, the results of the initial experiments with clownfish showed that the carbon dioxide had very few physical effects on the fish but had severe behavioral effects. What was not discussed in my video was the cause of these changes. GABAa is a well known inhibitory neurotransmitter. When GABAa bonds to a receptor it allows Cl- ions into the neuron which prevents it from firing. When carbon dioxide was added to seawater the GABAa inhibitor still able to bind to its receptor, but the distribution of ions inside the fish was altered. When the receptor site opens Cl- ions do not enter the cell but escape it instead. So when GABAa binds to a receptor in a clownfish in acidic water the neuron is excited rather than inhibited as it would be in an unaffected clownfish. We know that GABAa is being affected because the introduction of gabazine, a GABAa antagonist, results in normal fish behavior despite high carbon dioxide. Most aquatic animals use GABAa as an inhibitor so this effect is likely to be seen with fish and animals other than clownfish and damsels.

This diagram shows the GABA neurotransmitter bonding with its receptor site. The result is Cl- entering the cell. A cell with more carbon dioxide will experience Cl- exiting the cell

rather than entering it.

My experiment will differ from past experiments in that I will combine increased oceanacidification with increased temperature and salinity. All of these changes are expected to occurin the near future.

The Idea is to see if these other two variables affect the behavioral changes seen in carbonDioxide exposed fish. I will also be interested to see if they result in more changes in the fish’sstate of health than carbon dioxide alone did.

Rather than only doing a control Vs. experimental group analysis I will also do a before Vs. afteranalysis. This will provide more accurate results as to what changes occurred. To keep track ofwhich fish is which I took pictures of each fish from both its right and left side. I printed thesepictures in groups according to the Specific fish and then the experimental group that that fishwas a part of. Because of this design I was not able to keep the fish in groups of 6 as I hadoriginally planned because identifying individuals accurately would be nearly impossible. InsteadI planned to keep them in pairs and run the experiment 3 times with different fish each time.This design has the added benefit of showing repeatability to my results.

• Experimental Conditions:

I am considering “Normal” to be the average measured value ofthe great barrier reef, one of the most common places to findclownfish.

• Normal Seawater: pH 8.2• Past experiments tested: mainly pH 8.2, 7.8, 7.6• My Experiment: pH 7.8 (expected by 2100)

• Normal Salinity: 35 ppt (parts per trillion)• My experiment: 42 ppt

• Normal Temperature: 25 C• My Experiment: 28 C

To understand what variable any resulting variations from the originalexperiments are caused by I will need to test every combination of my3 variables. To do this I will need 7 experimental groups and onecontrol group.

The experimental combinations are as follows with the variable(s)written after a number being the one(s) to be altered:

1.) CO22.) Salinity3.)Temperature4.) CO2 + NA5.) CO2 + Temp6.) Temp + NA7.) CO2 + Temp + NA

Hypothesis• I expect that the same results seen in past tests with ocean

acidification will occur in all 4 tanks that I add carbon dioxide to. So I expect the results of the tests to be as follows:

Health testsWeight: No changeRespiration: No changeEating: No changeSensory TestsLateralization: The fish will no longer show this traitNet: The fish will swim towards the net rather than away from itFood: The fish will swim away from the food rather than towards it Tap test: The fish will always jump at the tap instead of stopping after afew taps

• I expect to see weight loss, a drop in eating, and quicker respiration in fish exposed to higher temperatures. I also expect that the tanks that have higher temperature combined with higher CO2 will see more dramatic changes in these health results than will the tank with high temperature only.

• I don’t expect to see any change in either behavior or health with the increase in salinity when it is alone or combined with the other variables. The reason I am testing it is because I want to see if the NA+ Ions affect the GABAa receptor/Cl- process at all. I am curious but I don’t expect it to cause any changes.

• Keep in mind that research has found some fish to be more tolerant of acidification than others. This means that most fish may be affected at a certain pH but others will show no changes. While this makes analyzing results a little more difficult, it does offer the possibility of selection in the fish’s natural habitat. It was not until a pH of 7.6 was reached that all fish were affected. At this point many fish had lost smell completely rather than following the opposite scent trail.

HealthWeight Test

• Weight: The weight of each fish will be measured. I expect that fish that are stressed for extended periods of time may experience weight loss while healthy fish will most likely increase in weight. Fish will be fed an excess of food at each feeding to insure that food availability does not affect weight gain/loss.

• I have not found previous research that monitored fish weight so I have nothing to compare this to.

SensoryTurkey Baster Tests

• Turkey Baster Test, part 1: Before experimentation, the fish were trained to expect food when a common turkey baster is lowered into the tank. As a result, the fish will bite the turkey baster whenever it enters the water. If the fish still exhibit this behavior after treatment then their long term memory, or the fish equivalent associations, are in tact.

• Turkey Baster Test, Part 2: a second turkey baster with a black tip will be introduced to the tank for the first time after variable exposure. Instead of food, this baster will shoot cold water towards the fish if they bite it. If, within a few days, the fish learn to bite the normal baster and avoid the baster with the black tip, then they will have shown the ability to form new associations.

SensoryT-Tests

• T-Test Part 1: All fish have what is called lateralization. This means that when faced with a decision as to turn left or right, an individual fish will choose either left or right the majority of the time and this behavior will remain constant for the fish over time. This could be compared to the human being left or right handed. However one side is not more common as it is with humans. The preference of each fish will be recorded using a "T" shaped tank before the experiment and then the fish will be tested again during the experiment to see if the treated fish will retain their lateralization or not.

SensoryT-Tests

• T-Test Part 2: A net, representing a predator, will be placed at one end of the "T" shaped tank. This will be repeated 6 times at random ends of the tank. Fish that wish to avoid being caught will swim opposite the net even if it defies their lateralization preference. After treatment some fish may loose the ability to accurately avoid the net.

T-Test Part 3: After not being fed for 24 hours food in a tea strainer (so that the fish can't eat it) will be placed at random ends of the Tee. If the fish turns towards the food despite its lateralization preference, then it retains both the instinct to get the food and the olfactory sense that allows it to smell the food.

• T-Test Part 3: After not being fed for 24 hours food in a tea strainer (so that the fish can't eat it) will be placed at random ends of the Tee. If the fish turns towards the food despite its lateralization preference, then it retains both the instinct to get the food and the olfactory sense that allows it to smell the food.

• This test worked for the practice fish that I tested it on before testing any of my experimental fish. However I had to replace the water in the T tank each time I ran the fish through so the process took over an hour. With 16 fish to test this was simply not practical.

SensoryUnused Tests-T Test Part 3

HealthUnused Tests-Respiration Test

• Respiration test: Fish breathe by pumping water through their gills, a process that is clearly visible to the naked eye. Respiration will be visually monitored for one minute to produce a breaths per minute rate. In general, a stressed fish will breathe either very quickly or very slowly while a healthy fish will lie somewhere in

between.

• This test was not completed because placing the fish in a confined area caused their respiration rate to increase so rapidly that any differences between healthy fish and unhealthy fish would have been impossible to determine

HealthUnused Tests-Eating Test

• Eating Test: After not being fed for 24 hours, each fish will be offered as much food as it will eat in one sitting. The mass of the food consumed will be closely measured by zeroing a scale with food and recording the absolute value of the mass of the food removed from the scale. Healthy fish will eat larger amounts than will unhealthy fish. The mass of food eaten will be compared to body mass of the individual fish rather than being directly compared to the amount of food eaten by other fish that may vary in size.

• This test was not used because when the fish were place in a confined space where their eating could be observed they refused to take food. This test worked quite successfully a few times for a fish that was alone in a tank but whenever 2 fish were separated they would become too stressed to accept food. I attempted leaving the fish alone for several minutes before offering food but they still would not try to eat it.

SensoryUnused Tests-Tap Test

• Each fish will be placed in a container with an automatic spinning weight that will hit the outside of the container every 10 seconds. Fish will jump when the weight hits the side of the tank. Over time they will remember that nothing bad happens after the weight hits the tank and stop reacting. A fish with no working short term memory will not be able to make this association and jump each time the weight hits the side of the container.

• This worked for a number of fish but many either never jumped at the sound or jumped every time during the pretest. This offered nothing to compare to after experimental conditions were initiated.

Results: Part 1• Due to the time constraints of high school, applying for college, and

other extracurricular activities I was only able to test one round of fish before having to present my findings. Thus my experiment will not be scientifically valid until I am able to run at least 6 fish per group (providing the experiment being run more than once) and show consistent results. As of right now I have placed 2 fish from each of the 8 sets of variables in experimental conditions and run them through the health and sensory tests both before experimentation and after 1 week of variable exposure.

• I also had a parasite get into my salt only tank. This killed one of the fish and the other had to be moved to a quarantine tank for medication. This

salt only tank was unable to be tested

• I will present the data that I have collected so far but keep in mind that it is not yet able to be accepted as scientific fact.

Weight Test-Results• Total: Average weight change was a loss of .76%• Tanks with CO2-lost .6% of their initial body weight on average, 21% more weight retained than the total

average. The CO2 only tank gained 1.125% of their body weight,

• Tanks with heat lost 1.27% of body weight, 67% more loss than the total average.

• Tanks with salt lost 1.1% of their body weight on average, 44.7% more than the total average. To calculate this I doubled the values for the CO2 + salt tank since CO2 appears to have no effect on the fish’s weight

• Control tank lost 3.65% on average. However one fish picked on the other constantly and the fish that was picked on was the one that lost an excessive amount of weight. This is a good example as to why this needs to be repeated as many times as possible to assure accurate results.

• Note: while most of the averages showed a drop in weight, many fish gained weight as well. There were also a few tanks that showed one fish gaining weight or remaining the same with the other fish loosing weight. This is likely because of the clown fish’s aggression in groups. One fish will become dominant (the eventual female) and pick on the other (male). This hierarchy can result in loss of weight from the stress of being picked on or because the dominant fish is more aggressive during feeding. To lean more about this visit the background on clownfish page.

• If this data were from a larger pool I would conclude that heat is detrimental to health and caused the fish to loose weight. I would not make a conclusion about salt without a salt only tank.

DataWeight DataControl Heat CO2 CO2 + Heat

CO2 + Salt Salt +Heat Salt + Heat + CO2

Fish #1Before: 2.51After:2.5

Fish #2Before: 2.45gAfter: 2.37g

Fish #1Before: 1.74After: 1.78

Fish #2Before: 2.38After:2.38

Fish #1Before:1.71gAfter:1.72g

Fish #2Before:2.39gAfter:2.43g

Fish #1Before: 2.25gAfter: 2.23g

Fish #2Before: 1.77gAfter:1.71g

Fish #1Before: 1.54gAfter:1.54g

Fish #2Before: 1.65gAfter: 1.64g

Fish #1Before: 2.31gAfter:2.26g

Fish #2Before:1.56gAfter:1.5g

Fish #1 before: 1.43after: 1.40

Fish #2Before: 1.98After: 1.98

Heat

Omitted

T-Test-Results• Control: 15% average change• 28.6% total change

• All Heat tanks average change: 27.5%, heat only: 35% • All CO2 tanks average change: 18.75%, CO2 only: 20%• All Salt tanks average change: 15%

• Notable: CO2 + Heat resulted in both fish having drastically altered lateralization with one fish’s lateralization being completely reversed. However, the CO2 + Heat + salt tank and the CO2 + salt showed little change. As is expected with such little data there appears to be some inconsistency.

DataT-Test-Percent of Time Turned Right

Control Heat CO2 CO2 + Heat

CO2 + Salt Salt +Heat Salt + Heat + CO2

Fish #1Before: 70% After: 80%

Fish #2Before: 50%After: 30%

Fish #1Before: 70% After: 10%

Fish #2Before: 60% After:70%

Fish #1Before: 70%After: 40%

Fish #2Before: 20%After: 10%

Fish #1Before: 60%After: 90%

Fish #2Before: 30%After: 80%

Fish #1Before: 80% After: 60%

Fish #2Before: 80% After: 80%

Fish #1Before: 30% After: 10%

Fish #2Before: 50%After:10%

Fish #1before: 70% after: 60%

Fish #2Before: 90%After: 90%

Heat

Omitted

10% Change

20% Change

60% Change

10% Change

30% Change

10% Change

30% Change

50% Change

20% Change

No Change

20% Change

40% Change

No Change

10% Change

T-Test

I could find no consistency in the T-test turn results so I can not make any conclusions until

I repeat my experiment.

DataT-Test-Percent of Time Turned Away From Net

Control Heat CO2 CO2 + Heat

CO2 + Salt Salt +Heat Salt + Heat + CO2

Fish #1Before: 66% After: 50%

Fish #2Before: 83%After: 66%

Fish #1Before: 100% After: 100%

Fish #2Before: 83% After: 83%

Fish #1Before: 100%After: 66%

Fish #2Before: 50%After: 83%

Fish #1Before: 66%After: 66%

Fish #2Before: 100%After: 33%

Fish #1Before: 66% After: 50%

Fish #2Before: 100% After: 83%

Fish #1Before: 100% After: 66%

Fish #2Before: 100%After: 100%

Fish #1before: 83% after: 66%

Fish #2Before: 50%After: 83%

Heat

Omitted

-16% Change

-17% Change

0% Change

0% Change

-33% Change

+33% Change

-67% Change

-16% Change

-17% Change

-33% Change

0% Change

+33% Change

-17% Change

-0% Change

Turkey baster tests #1 and #2

• All fish were attracted to the original turkey baster after testing had started. I tested them 4 days into experimental conditions and again 7 days into experimentation with the same results both times.

• All fish were afraid to come up to the turkey baster with the black tip when it was put into the water initially. I tested them at 5 days and again at 7. At the 7 day test I added food to the baster and while the fish came closer to it because they saw the food they were still wary of it.

• No variation in results was seen for any fish

Conclusion• Although very little can be determined from the data collected so far I found it more

important that I learned a lot about the process of scientific research.

• Improvements that could have been made:

• More tests could be run on the fish. So many tests that I planned to run were not run, therefore; very few sets of data were left to analyze changes in the fish.

• I would use bigger tanks so that I could keep more than 2 fish in them. Even though I used 30 gallon sumps the 10 gallon main tanks I used did not allow for more than 2 fish without deaths from fighting. Perhaps a good solution to this would be to use tank dividers.

• Higher quality heaters and pH controllers would have offered more accurate experimental conditions. The funding for these expensive items was not there.

• I would like to run experimental conditions longer next time and test the fish several times during this period to find out exactly how long after conditions are initiated that the fish begin to be affected.

Thank you…

• http://www.newscientist.com/article/dn21355-carbon-dioxide-encourages-risky-behaviour-in-clownfish.html?DCMP=OTC-rss&nsref=deep-sea

• http://wottsupwiththat.com/2012/01/18/co2-increases-to-make-drunken-clownfish/

• http://www.newscientist.com/article/dn21355-carbon-dioxide-encourages-risky-behaviour-in-clownfish.html