Torrey Centeno and Emily Gallagher Period 4 2017 Reproduction Behaviors in Lined Seahorses, Maroon Clownfish, and False Percula Clownfish Torrey Centeno and Emily Gallagher Period 4 2017

Focus Examine reproduction behaviors of clownfish and seahorses Sustain population of clownfish larvae Advantage of different species of clownfish and ability to create rotifer cultures

False Percula Clownfish Amphiprion Ocellaris Tank 11 Bright orange color, varies less from males to females Territorial Lay eggs every 10-14 days Eggs hatch after ~8 days -False Perculas held in Tank 11 -Well-known for bright orange color, males and females have very similar coloring -Territorial -Clutch averages Tullock, John H. Clownfishes and Sea Anemones. 1998.

Larval Stage -We came across this on nearly every website we visited: clownfish larvae are HIGHLY vulnerable -Very high mortality rate -Because they can’t consume normal fish food, the larvae must feed ideally on a diet of rotifers (microscopic living algae)

Maroon Clownfish Premnas Biaculeatus Three white body bars with a color ranging from orange in males to dark red and brown in females. Living with other species in Tank B Aggressive 10-14 days between clutches 8 days to hatch -Dark red color -In Tank B, the maroons were integrated with other species of fish and invertebrates -Very aggressive (more than False Perculas) -Without any outside influences, they have the same clutch averages as False Perculas -With the research showing that clutch averages were the same, we could use the tank environment differences to structure our research and give us a method to compare behaviors of similar organisms in different environments Wilkerson, Joyce D. Clownfishes: A Guide to Their Captive Care, Breeding, and Natural History. 1998.

Lined Seahorse Hippocampus erectus Light brown color with banded markings around the body Males larger in size and have a brood pouch on abdomen to hold eggs Complex courtship Color changes Dependent on photoperiod -We discovered as we did further research and observation that the males were the larger seahorses with pouches on stomachs to hold eggs -Courtship ritual involves dancing (often in the morning) -organisms will mimic each other’s swimming patterns and sometimes undergo a color change -Seahorses rely on the sun to tell them when to mate, photoperiod is significant

Research Questions Does the species of clownfish affect the reproduction behavior/survival rate of larvae? Are there certain rocks/anemones more conducive to breeding? Does tank environment affect reproduction behavior? Will isolating one pair of seahorses help the male accept the eggs from the female? Can we maintain a healthy rotifer culture to feed the larval clownfish and seahorse fry? -Take research about species and tank environment into consideration and examine both of these factors -Maroons had rock, perculas had flowerpot -Because courtship ritual is so intricate, will presence of three seahorses disrupt mating pattern -Once we had eggs that hatched, we needed to sustain the population with rotifer culture Wilkerson, Joyce D. Clownfishes: A Guide to Their Captive Care, Breeding, and Natural History. 1998. Ruiz, José. "Breeding Seahorses." Tropical Fish Hobbyist Magazine. N.p., Apr. 2010. Web. <http://www.tfhmagazine.com/details/articles/breeding-seahorses.htm>. Lourie, Sara A. Seahorses: A Life-Size Guide to Every Species. 2016.

Background Research Ruiz, José. "Breeding Seahorses." Tropical Fish Hobbyist Magazine. 2010. Web. This article provided a complete description of the process of breeding seahorses, from mating to rearing. The information about specific elements of the tank environment is helpful to know so that we can make the correct adjustments. Wilkerson, Joyce D. Clownfishes: A Guide to Their Captive Care, Breeding, and Natural History. 1998. This source also helped to clarify our identifications of our clownfish species. However, what really separated this source from others is that it provided in depth knowledge about clownfish breeding and the reproductive process. It also gave specific details about each of our species of clownfish and highlighted their differences from each other.

Background Research Lourie, Sara A. Seahorses: A Life-Size Guide to Every Species. 2016. This book was very helpful when trying to identify the type of species within the tank. It listed many facts about the habitat of our species of seahorse and gave specific information about its diet and behavioral traits as well. It also provided numerous information about the mating process of seahorses and helped us formulate our questions about factors to change to help coerce the seahorses into mating. Tullock, John H. Clownfishes and Sea Anemones. 1998. This source helped in clarifying our identifications of the clownfish species. It also provided additional information on their reproduction process such as details about their mating and the laying of the eggs. It also gave basic information for taking care of reproducing clownfish in a tank setting.

Hypothesis Clownfish: By creating a new way to transport the eggs to a separate tank while maintaining the proper conditions and maintaining a healthy rotifer culture, the clownfish larvae will have a better chance of survival. Seahorses: By manipulating the ambient temperature and lighting, and possibly isolating a pair of seahorses, this will simulate natural mating and induce the seahorses to reproduce. -Improve mortality rate and vulnerability of clownfish larvae by changing environment and diet (Joyce D. Wilkerson, Clownfishes: A Guide to their Captive Care, Breeding, and Natural History) -Changing seahorse environment to mimic a more natural setting in which they would be more comfortable reproducing (Jose Ruiz, Tropical Hobbyist Magazine)

Methods Updated data sheets Recorded temperature, salinity, behavior Swimming, cleaning eggs, courting, expecting Tanks B, 10, 11 Location of Eggs Attempted rotifer culture Used refractometers Carboys Algal plates Proline A & B Pipettes Followed instructions created by Erin Ocana and Dominique David

Clownfish Data Sheet

Seahorse Data Sheet

Early Larvae Support Moved flowerpot with eggs attached to separate, smaller tank Set up aeration system to mimic male cleaning eggs No filtration system -Set up new tank for eggs laid in tank 11 -Bubbler mimics behavior of male clownfish as he aerates the eggs -No filter, less disrupting for when the larvae did hatch

False Perculas laid more frequently Isolated in tank Only 2 observed clutches of Maroon eggs According to research, clutch data shouldn’t vary between species Does the species of clownfish affect the reproduction behavior/survival rate of larvae? o Maroon clownfish had drastically different observed clutch data, but based on research this was most likely because of their environment and not the species o Only hatched false percula larvae, had no basis of comparison for maroon clownfish larvae o Species of clownfish doesn’t affect larvae as much as tank environment does Wilkerson, Joyce D. Clownfishes: A Guide to Their Captive Care, Breeding, and Natural History. 1998.

Irregular data for Maroon clownfish Should be about 10-14 days between clutches Very different tank environment -Irregularity in the data -Not only did they lay eggs less times, but the time in between the sets of eggs was much higher Are there certain rocks/anemones more conducive to breeding? o Flowerpot- larger surface to breed on instead of exposed rock Does tank environment affect reproduction behavior? o Yes! The maroon clownfish were very limited in their tank environment due to other species o Had one area to constantly defend instead of having free range of a tank o False perculas didn’t have threats from other species Wilkerson, Joyce D. Clownfishes: A Guide to Their Captive Care, Breeding, and Natural History. 1998.

Male seahorse died on 11/23 Other male/female pair already familiar with courtship Increased after second male was taken out of environment (Ruiz 2010) Eggs observed in March- one of only recorded broods in Lovett program Will isolating one pair of seahorses help the male accept the eggs from the female? o Yes! Once the male seahorse died, the remaining male and female engaged in courtship and laid eggs o Courtship had been observed with second male present, but had never resulted in eggs Ruiz, José. "Breeding Seahorses." Tropical Fish Hobbyist Magazine. N.p., Apr. 2010. Web. <http://www.tfhmagazine.com/details/articles/breeding-seahorses.htm>. Lourie, Sara A. Seahorses: A Life-Size Guide to Every Species. 2016.

Initial larval stage (Wilkerson 1998) Higuera 2006 -One of the successes of this project was the second clutch of eggs that we hatched -Able to keep larvae alive longer than had ever been previously recorded at Lovett -Had 2 to 3 organisms out of ~25 that actually transitioned to the juvenile stage -If we had had a rotifer culture, fish would have survived even longer

Eggs Sighted on Feb. 28 Lourie. 2016 Eggs Sighted on Feb. 28 Research found from Lourie. Seahorse Lifesize Guide to Every Species 2016 helped confirm outcome/hypothesis Probably would have actually taken up eggs if lighting/temperature adjusted Eggs Sighted on Feb. 28 Lourie. 2016 Eggs Sighted on Feb. 28 Lourie. 2016

Challenges Rotifer culture- not able to sustain culture Expired algae plates Successful early hatching, but didn’t continue this process with later hatches because of rotifer problems Focus of research shifted to breeding habits and away from raising hatched organisms Can we maintain a healthy rotifer culture to feed the larval clownfish and seahorse fry? o We don’t know. We were limited by time and equipment that didn’t function properly, so we were never able to get the rotifer culture up and running -One of the successes of this project was the second clutch of eggs that we hatched -Able to keep larvae alive longer than had ever been previously recorded at Lovett -Had 2 to 3 organisms that actually transitioned to the juvenille stage -Because culture was not sustained, we had to create a new focus for project

Hypothesis Reflection By creating a new way to transport the eggs to a separate tank while maintaining the proper conditions and maintaining a healthy rotifer culture, the clownfish larvae will have a better chance of survival. Partly proven New larvae tank setup increased survival rate of organisms Not able to successfully maintain rotifer culture, but this most likely would have further increased survival rate of fish -Done differently: kept up with rotifer culture, recorded data for survival rate of larvae

Not able to adjust temperature or lighting By manipulating the ambient temperature and lighting, and possibly isolating a pair of seahorses, this will simulate natural mating and induce the seahorses to reproduce. Partly proven Not able to adjust temperature or lighting Isolated pair definitely more conducive to breeding -Done differently: kept closer observation of male and eggs when closer to hatching

Conclusions Hypotheses have potential to be fully proven with more research Data about reproduction patterns can help guide further research, test different variables with maroon clownfish Fish reproduction is a complex process Can’t replicate certain elements found in natural environment, breeding in captivity requires more work and attention to detail -Work and observations we completed set up the project to be improved upon next year -Since we know that clownfish reproduce more successfully if isolated, potential tests could include isolating the maroon clownfish -Juveniles can be raised to adulthood with proper diet

Thanks and Be Sure to Visit Our Website! https://lovettfishbreeding.wikispaces.com/

Sources Brough, Clarice. "Maroon Clownfish." Animal World. N.p., n.d. Web. 04 May 2017. Higuera, J. 2006. "Premnas biaculeatus" (On-line), Animal Diversity Web. Accessed May 04, 2017 at http://animaldiversity.org/accounts/Premnas_biaculeatus/ Lourie, Sara A. Seahorses: A Life-Size Guide to Every Species. 2016. Michael, Scott W. Marine Fishes: 500+ Essential to Know Aquarium Species. 1999. Ruiz, José. "Breeding Seahorses." Tropical Fish Hobbyist Magazine. N.p., Apr. 2010. Web. <http://www.tfhmagazine.com/details/articles/breeding-seahorses.htm>. "Seahorse Reproduction." Seahorse Worlds, www.seahorseworlds.com/seahorse-reproduction/. Tullock, John H. Clownfishes and Sea Anemones. 1998. Wilkerson, Joyce D. Clownfishes: A Guide to Their Captive Care, Breeding, and Natural History. 1998. Nemo pic: https://www.google.com/