1. Reproduction of Amphiprion ocellaris and Amphiprion clarkii in relation to behavior and number of tank mates Kelly Williams & Maggie Wolters 2011-2012

2. How We Got Started… The experiment is designed to observe the reproductive behaviors of clownfish. It will test whether or not the clownfish are more likely to reproduce in a solitary environment versus in a tank with multiple organisms at constant temperatures, salinity, and lighting. While observing breeding behaviors and activity, the experiment will also allow to test for preferable surfaces during spawning. Data will be recorded based on everyday observations. It is hypothesized that clownfish are more likely to breed in a solitary tank, spawn on flat surfaces at optimum conditions for Amphiprion ocellaris and Amphiprion clarkii.

3. Proposal Background Initial Hypothesis – Although the time period that the pairs have been mated is varied, if both tanks are at optimum conditions, then the solitary Amphiprion clarkii pair in tank C-2 is more likely to reproduce on the tile and be less aggressive compared to the Amphiprion ocellaris pair in tank 90 that will produce on a flat rock. Experiment/Methods: – Everyday observations of behaviors of the clownfish and tank mates were recorded

4. Question 1 What are the differences in clownfish behavior between the solitary female Amphiprion clarkii versus the mated female Amphiprion ocellaris both normally and while breeding?

5. Q1: Results – October/November Observations – Tank 90: Female more active than male, fidgety – possibly caused by anemone movement Breeding: male aerated eggs/stayed beside host anemone, while female remained active Video 2 – Tank C-2: Female hiding in Xenia, fidgety, inactive (male not present) Video 1

6. Q1: Results – December/January Observations – Tank 90: Female roamed around tank while male remained close to or inside host anemone. When swimming together, the pair follows each other, especially around feeding time Breeding: Male aerated the eggs so remained very close anemone while female was more active, but stayed fairly nearby. – Tank C-2: Female remains close to Xenia, while the new male is relatively active (opposite of tank 90) *During the later months, we realized that the male and female A. clarkii switched swimming patterns

7. Tank 90 Tank C-2

8. Q1: Results - February Observations – Tank 90: Male and female A.ocellaris less interactive. Female more active about the tank while the male is stationary near the host anemone (occurs more in the afternoon). Anemone movement possible reason for different behavior – Tank C-2: More interaction seen between the male and female A. clarkii. Female now less fidgety and more active around the tank, while the male seems to stay near the Xenia. This is similar the the behaviors of the A. ocellaris in tank 90. Male has grown in size Video 3

9. Q1: Results - March Observations – Tank 90: More interaction between the male and female. They tended to follow each other more and stay close to the host anemone. – Tank C-2: No signs of breeding; however, the male and female clarkii have become even more interactive than in the previous month. The female is less fidgety and the male is more stationary in front or above the Xenia.

10. Question 2 Do clownfish prefer a tile or a flat rock for spawning?

11. Q2: Results – October/November Observations – Tile was not in tank 90 until December/January – Tank 90: Clownfish laid eggs on a rock under the host anemone – Tank C-2: No eggs present (no male)

12. Q2: Results – December/January Observations – Tank 90: Tile added to the center/front part of tank between host anemone and live rock. It was later moved to a location further back in the tank near live rock. The clownfish did not lay eggs. – Tank C-2: No tile present, and no eggs laid.

13. Q2: Results - February Observations – Tank 90: The tile is still present; however, it has not been effective considering the clownfish did not lay eggs during this month on neither the tile nor a rock. – Tank C-2: No tile present and no eggs were laid

14. Q2: Results - March Observations – Tank 90: The tile is still present in tank, but no eggs were laid this month. Same as previous month (February) – Tank C-2: There is still no tile in this tank and no eggs were laid on either surfaces.

15. Question 3 Are the clownfish that are isolated in tank C-2 more likely to breed compared to the clownfish living with other organisms in tank 90? Male clarkii in C-2

16. Q3: Results – October/November Observations – Tank 90: Eggs were laid on the rock in late October; however, because male remained close to the eggs, the tank mates did not bother or show aggressive behavior toward the clownfish. – Tank C-2: No male present, so no eggs were possible.

17. Q3: Results – December/January Observations – Tank 90: Eggs were laid in late December in the back of the tank. During feeding time, the clownfish appeared more aggressive toward their tank mates when they drew near to their eggs. – Tank C-2: No signs of breeding and no eggs were laid.

18. Q3: Results - February Observations – Tank 90: Considering there were no signs of breeding, no eggs were laid this month. Possible reasons were inconsistent lighting and algae growth (poor conditions) – Tank C-2: No breeding behaviors recorded and no eggs were laid. *No comparison can be made.

19. Q3: Results - March Observations – Tank 90: More interaction between the male and female ocellaris; however, no breeding behaviors were recorded, therefore no eggs were laid. – Tank C-2: No eggs laid this month. *Cannot make a comparison considering no eggs were laid in either months.

20. Question 4 What are specific aggressive behaviors that occur during clownfish breeding?

21. Q4: Results – October/November Observations – Tank 90: No aggressive behaviors exhibited by clownfish or tank mates were observed – Tank C-2: No other tank mates present around female

22. Q4: Results – December/January Observations – Tank 90: No aggressive behaviors were recorded except for during feeding time. The Regal Tang would sometimes get too close to the eggs and the female would chase him off by nipping. – Tank C-2: The clownfish pair did not show any signs of aggression towards each other and they had no other tank mates.

23. Q4: Results - February Observations – Tank 90: More aggression behaviors were recorded this month. The regal tang has been more aggressive towards the ocellaris and other tank mates. This has created greater stress for the clownfish. Video 4 – Tank C-2: The clarkii pair did not show any signs of aggression towards each other.

24. Q4: Results - March Observations – Tank 90: Less aggressive behavior from other tank mates seen this month. The clownfish pair was not aggressive towards each other either. – Tank C-2: No aggression behaviors between the clarkii pair were recorded this month.

25. Question 5 Will the pair that has been mated the longest in tank 90 be more likely to breed more than the new clownfish pair in tank C-2?

26. Q5: Results – October/November Observations – At this point, the clownfish pair in tank 90 had obviously bred more because tank C-2 only had a female – We believed at this time that following the addition of a male A. clarkii, the pair in tank C-2 would be more likely to breed

27. Q5: Results – December/January Observations – Tank 90: Once again, the clownfish in tank 90 have bred more than the other clownfish pair – Tank C-2: Since the addition of another A. clarkii, the two clownfish have no interacted very much. Possible explanations: Age difference, still adapting to each other, or not both in symbiosis with the Xenia

28. Q5: Results - February Observations – Tank 90: In previous months the ocellaris have bred and laid eggs but they have not this month. – Tank C-2: No breeding signs have occurred between the clarkii pair. The clownfish are still new to each other *Neither clownfish pair bred this month

29. Q5: Results – March Observations – Tank 90: Although the ocellaris pair has interacted more this month, no breeding behaviors or laid eggs were recorded. Possibility we did not see them because in April eggs were found behind a rock. – Tank C-2: No breeding occurred this month *Neither clownfish pair bred this month

30. October/November Ethogram

31. December/January Ethogram

32. February Ethogram

33. March Ethogram

34. Daily/Monthly Successes Currently witnessing the clownfish pair in tank 90 aerating eggs again behind live rock (third time) Algae growth is maintained Corals have remained consistently healthy over the entire year Interaction between the male and female A. clarkii

35. Daily/Monthly Challenges Algae growth (peppermint shrimp and crabs helped maintain healthy conditions, as well as removal by hand) Inconsistent lighting in tank 90 (purchased new lights in April) Did not purchase another A. clarkii until January for tank C-2 The death of the nudibranch in anemone (released toxins)

36. Other Observations Algae growth in both tanks Tile in tank 90 Age difference between clownfish pairs Female A. clarkii became less fidgety with addition of new male A. clarkii Activity of the anemones in tank 90

37. Anemone Movement

38. Conclusion Rejection of hypothesis – Although the time period that the pairs have been mated is varied, if both tanks are at optimum conditions, then the solitary Amphiprion clarkii pair in tank C-2 is more likely to reproduce on the tile and be less aggressive compared to the Amphiprion ocellaris pair in tank 90 that will produce on a flat rock. Why? – The Amphiprion ocellaris in tank 90 laid eggs 3 times while the Amphiprion clarkii did not breed once Alternative explanations – Age difference – Limited time together during this project (A. clarkii) – Algae growth – A. clarkii pair prefer tile? – A. ocellaris not really bothered by other tank mates in tank 90 during breeding