Newbie Research Tank

Background research

Protandrous hermaphroditic colony formation of Amphiprion ocellaris anemonefish triggered by hormonal and behavioral cues background research​

Anemonefish are saltwater, tropical fish that are found in the Pacific Ocean, from Australian, to Hawaiian, to Asian waters. Clownfish and anemonefish are the same animal. The name clownfish comes from the fast, jerky movements the anemonefish make when they swim. The fish look like they are the playing, or ‘clowning around’. Anemonefish in the wild are part of a symbiotic relationship with a sea anemone. The clownfish receive protection from predatory animals by hiding in the anemone’s stinging tentacles, and the clownfish protect their home by chasing away the fish that would like to eat the anemone’s tentacles (Newcomb & Fink, 2004). The anemonefish are not harmed by the nematocysts, or stinging cells of the anemones tentacles, because the fish forms a unique type of mucus that protects the neuro-poison from the nematocysts from reaching the fish. Other fish, that do not form the protective mucus barrier, are paralyzed when they are stung by the anemone (MarineBio.org, 2009).
Amphiprion ocellaris anemonefish, also commonly know as false percula clownfish, common clownfish or false anemonefish, are closely related to A. percula, or true percula clownfish. All clownfish are protandrous hermaphrodites. Protandry is the sex-change from male to female (Devlin & Nagahuma, 2002). Three other types of hermaphroditism are common in the fish world. There is protogyny, or sex change from female to male, and simultaneous hermaphroditism, where the organism is both sexes at once. There is also bi-directional hermaphroditism, where the organism changes sex several times throughout its lifecycle (Miura, Nakamura, Kobayashi, Piferrer, & Nakamura, 2007).
All species of clownfish form a protandrous colony consisting of one dominant female, one mature male, and several immature males (MarineBio.org, 2009). If the females dies, or is removed from the colony, the mature male will become the female and one of the immature males develops into the mature male. The female is in charge of the group, and she, together with the mature male, prevents the immature males from changing sex (MarineBio.org, 2009).
Many triggers have been found for sex reversal in ichthyology. These include environmental factors, genetics, and biological triggers (Devlin & Nagahuma, 2002). Genetics play a role in sex differentiation because only fish with the genes for hermaphroditism will change sex without outside interference. Environmental factors are part of sex differentiation because they often decide when the fish will change sex. With anemonefish, the sex change occurs after the dominant female is removed from the colony. Biological factors include the behavior of the fish within the colony of anemonefish (Devlin & Nagahuma, 2002).
Hormones have also been shown to have an impact of the sex differentiation of fish. When fish have higher levels of estrogen, the fish change into females. When the levels of estrogen are lower, the fish are males. Testosterone also plays a role in the maturation of the male anemonefish in the colony. The mature male anemonefish will have a higher level of testosterone that an immature male. Enzymes that aid in the absorption of the hormones, such as aromatase (Guiguen, Fostier, Piferrer, & Chang, 2009), GnHRa (Shin, An, Park, Jeong, & Choi, 2009), and enzyme E2 (Gardner, Anderson, Place, Dixon, & Elizur, 2004) factor into sex differentiation as well. These enzymes have been shown to have an effect on sex differentiation in other species of fish, but not anemonefish. Also, the research has been done on the impact of the enzymes on the sex change of the fish, not on how
My research is going to focus not on how the fish change sex, but how the dominant female and the mature male are able to prevent the immature males from maturing, and how the female prevents the mature male from becoming female. The research will be testing environmental factors, such as testing to see if the dominant pair puts out a hormone or enzyme that prevents the immature male from producing testosterone and becoming a mature male, and if the female secretes a hormone or enzyme that prevents the mature male from producing or absorbing estrogen. I will also test the behavioral factors of sex change, by placing the female in a separate tank where the other fish are still able to see her, and observe whether the behavior of the colony changes as if she were dead, or if the dynamics of the colony remain stable. Both will be tested separately, but results will be compared. The research will help to answer whether hormones prevent the immature fish from changing sex, if behavior of the dominant pair prevents immature males from changing sex, or whether it is a combination of both, or neither.

 

Sounds like you've done some good research. Personnally I think that it isn't triggered by hormones because the fish would have to produce a lot of hormones/enzymes to prevent sex change in the other males when you take into account changing currents, strong currents and dilution by a very vast ocean.

Will this be a closed system? If it is, then it will be difficult to work out if hormones are responsible because the hormones will be a lot more concentrated in a closed small volume of water than in the ocean. Unless you set up a tank with males and a female under flow through conditions, where new sea water is pumped into the tank and then leaves the tank to a drain, to stop any hormones from building up.

 

That's my thoughts as well. Also, I'm pretty sure that it would be unique among vertebrates to be excreteing hormones into the environment but then again protangenous hermaphrodism isn't exactly common either.

I also guess that hormones present in the environment in sufficient concentration to affect multiple fish would also affect the fish secreting them.

I'll remain suscribed to this thread and hope that in a year or 2 we'll know your results.

 

here's the ED:
Protandrous hermaphroditic colony formation of Amphiprion ocellaris anemonefish triggered by hormonal and behavioral cues
Problem: How does the mature female and male within the colony prevent the immature male fish from changing sex?
Hypothesis: Without the stimuli from the female fish, the mature male fish will change to be a female, and an immature male will mature into a mature male. If the males are prevented from changing sex, then the female must be preventing the sex change of the mature male into a female.
Independent variable: hormonal and behavioral influence of the mature female anemonefish
Dependent variable: the final sex of the immature fish in the colony
Constants: all of the fish are kept in the same environment within the tanks
Control: the initial sex of the fish in the colony
Repeated trials: several sample of the same water can be tested, and removing the mature female fish from the colony would allow the mature male to become the new female for several trials
Materials:
50-55 gallon tank aquarium stand Styrofoam
over-the-back filter aquarium heater protein skimmer
aquarium thermometer power heads saltwater mix
rock substrate refractometer
air stones and pump fishnets new 5 gallon buckets
20 juvenile (6months -1 year) A. ocellaris anemonefish model female anemonefish
fishing line ES (total estrogen) ELISA test kit
plate reader 11-ketotestosterone ELISA test kit





Procedure:
1. Set up marine 50-55 gallon marine aquarium.
a. Set up the tank stand where it is level, not in direct sunlight, and in an area where the floor can support the weight of the full tank. Place a layer of Styrofoam under the tank to help with the fit of the tank
b. Wash the aquarium with freshwater and place on tank stand
c. Set up power heads, biological filter, over the back filter, protein skimmer, air pump, and air stones, then run the setup with freshwater for a day to test for leaks
d. Set the heater to raise the tank to desired temperature,74°F-80°F(23°C-27°C), while testing the tank setup
e. Add saltwater mix to tank until desired specific gravity, 1.020-1.025, is reached
f. Let the tank run for 24 hours to test for leaks and seepage, as well as to check how well the different components of the system are working
g. Add substrate and arrange rocks to form a stable pattern of hiding areas for the fish, to promote their natural behavior
h. Place boiled shrimp in that tank to begin the nitrogen cycle.
i. The nitrogen cycle within the tank could take 6-8 weeks, and it is over when the ammonia and nitrite levels read 0 when they are tested
j. Fish will be added after the tank has finished cycling, 1-2 at a time. After each installment of fish, the nitrogen cycle will begin again, and no more fish will be added until the readings for ammonia and nitrite are once again 0, after about 2 weeks. Fish will be added until there are 20 test subjects
k. Fish will be fed once a day on a mixture of premade flake food and meaty foods such as cooked seafood that has been cut into small pieces. Condition of fish will be observed daily at feeding. Temperature will be measured daily to be 22-26ºC and recorded in the lab notebook to watch for trends
l. Specific gravity near 1.023 with no more than 0.001 units, pH between 8.0-8.4, ammonia at 0.0ppm, nitrite at 0.0ppm, and nitrate at less than 30ppm will be measured once a week, recorded in the lab notebook to watch for trends, and make corrections. Consistency is more important than the absolute value of the readings.
m. After the tank has been cycled, water changes will be made when nitrate levels reach 30ppm to bring down the level of nitrates. Approximately 25% of the water will be changed once a month.
2. The fish will be fed a varied diet of flake food and fresh, meaty food once a day for optimum conditions
3. Place the model female anemonefish in saltwater, and test water sample to ensure the materials of the model do not leach into the water. See appendix A for procedure for the 11-ketotestosterone ELISA test kit.
4. Use fishing line to attach the model female clownfish to the tank so it float stays at midlevel in the tank
5. Observe the anemonefish and the model to see if the model is able to prevent an immature male from maturing into a mature male and/or a female
6. If the model clownfish does not prevent sex change in the colony, water will be tested using the 11-ketotestosterone ELISA and ES (total estrogen) ELISA test kits. A plate reader is used for reading the plates of the test kit.
7.
A. ocellaris anemonefish will be used because they are one of the few organisms that are readily available and show protandrous hermaphroditism. Using invertebrates that exhibit protandrous hermaphroditism, such as shrimp from the family Pandalidae, for the test subjects is not feasible because they are far more complicated to care for than A. ocellaris, and they are mainly rare and endangered species. Those organisms also do not exhibit colony formation and the prevention of sex change found in A. ocellaris colonies. A. ocellaris is one of the most hardy aquarium fish, is readily available, and is often recommended for beginner aquarists because of its forgiving nature in an aquarium.