Stripping Clowns: Induced Meristic Changes in Common Clownfish (Amphiprion ocellaris) Pigmentation des poissons-clown : induction de changements méristiques chez Amphiprion ocellaris Scott E. CLEMENT1, Joseph H. LICHTENBERT2, Christopher C. KOHLER1 1 Fisheries and Illinois Aquaculture Center, Southern Illinois University at Carbondale, Carbondale, IL 62901, USA 2 Reef Propagations Inc., Hoffman Estates, IL 60194 ABSTRACT Thyroid hormones are known to induce metamorphosis in larval fish, but few studies have examined the changes in pigment alongside the musculoskeletal development. A study was undertaken to examine the effects of exogenous T3 (triiodo-L-thyronine) on the development and pigmentation of larval common clownfish (Amphiprion ocellaris). Larvae were collected after hatching and split into two groups. One group was immersed in artificial seawater with 2 mg/L T3, the other in artificial seawater without hormone, for one hour. Both groups were transferred to separate tanks within a common rearing system, and raised through the development of juvenile pigmentation. Significant differences in dorsal spine numbers became evident by day 14. The control group developed all three white bars, but the T3 group only developed partial or complete tail bars, with a few individuals never showing any bars. These results indicate a major role for T3 in larval clownfish development. RÉSUMÉ Les hormones thyroïdiennes sont connues pour déclencher la métamorphose des larves de poissons, mais peu d’études ont examiné les changements de la pigmentation en même temps que le développement des muscles et du squelette. Une étude a entrepris d’examiner les effets du T3 (triido-L-thyronine) exogène sur le développement et la pigmentation des larves de poissons-clown communs (Amphiprion ocellaris). Les larves furent collectées après éclosion et séparées en deux groupes. Le premier groupe fut immergé dans de l’eau de mer artificielle contenant 2mg/l de T3, le second dans de l’eau de mer artificielle ne contenant pas d’hormones, tous deux pendant une heure. Les deux groupes furent alors transférés dans des réservoirs séparés, à l’intérieur d’un même système d’élevage, et élevés selon le développement de la pigmentation juvénile. Des différences significatives dans la numération de l’épine dorsale apparurent clairement à partir du quatorzième jour. Le groupe de contrôle présentait bien trois rayures blanches, tandis que seul le groupe T3 présentait des rayures partielles ou complètes sur la queue, avec quelques spécimens ne présentant pas du tout de rayures à aucun moment. Ces résultats indiquent que le T3 tient un rôle majeur dans le développement des larves de poissons-clown. Bulletin de l’Institut océanographique, Monaco, n° spécial 20, fascicule 1 (2001) INTRODUCTION It has long been known that thyroid hormones are involved in growth, maturation, and metamorphosis. A classic experiment in endocrinology involves increasing the rate of metamorphosis in tadpoles by immersion in triiodo-Lthyronine (T3) or L-thyroxine (T4). Metamorphosis in fish has also been shown to be driven by thyroid hormones, yet in most studies little quantification of the changes in pigmentation have been made. Reddy and Lam (1992) and Brown and Kim (1995) showed changes in larval fish pigmentation after immersion in T3 or T4. The objective of this research was to examine the effects of exogenous T3 on the pigmentation and development of larval common clownfish (Amphiprion ocellaris). METHODS Amphiprion ocellaris larvae were collected in a plexiglass container once hatching was completed one hour after lights were turned off. The container was moved to a lighted room, where the contents were split into two groups of approximately 350 larvae each. A 125 ml aliquot of a 10 mg/L T3 stock solution was added to the water of one group to reduce the concentration to 2 mg/L. Both groups were kept in the immersion for one hour, then transferred to separate tanks within a common larval rearing system. After transfer, a lighting cycle of 15.5 L: 8.5 D was initiated. Standard clownfish larval rearing methods were used to raise both groups through completion of metamorphosis. RESULTS Differences were noticeable between the two groups within the first 48 hours. The T3 group appeared orange, while the control group exhibited silver color characteristic of larvae. By day 6 the T3 group was 25-33% smaller than the control group. Metamorphosis from larval to juvenile behavior occurred at roughly day 10 for both groups, but a small percentage of the hormone treated group swam head up at a 45° angle indicating a lack of swimbladder inflation. Only the control group developed head bars at that time. Significant differences in dorsal fin spines became apparent by day 14. By day 17, 95% of the control population had both head bars and middle bars while the T3 group showed little evidence of any barring. By day 30, the control group had all three bars. Figure 1 illustrates the differences between treated and control fish at several months of age. The top two fish were from the T3 group and show little to no white bars, and few if any first dorsal spines. A dorsal inflection of the pectoral fins can also be seen, but may also be due to a lack of some pectoral rays. The bottom two fish were from the control group and show all three complete white bars, and all 11 first dorsal spines. Eventually the T3 group developed complete or partial tail bars, though some individuals never developed any bars. Figure 2 illustrates some of the variation in the T3 treated fish. Note the lack of head and middle bars, dorsal spines, and even some of the black pigment Bulletin de l’Institut océanographique, Monaco, n° spécial 20, fascicule 1 (2001) that should have been present on the end of the pectoral and anal fins. Some anal fin rays may have been missing from the treatment group, however this was not quantified. By day 60, no differences in size between the groups were evident. # Bars # Dorsal Spines 12.0 10.0 8.0 6.0 4.0 2.0 0.0 Control T3 Mean differences in the number of dorsal spines on juvenile A. ocellaris after control and T3 treatment of larvae. N = 25 fish from each group. Control and treatment groups were significantly different (p < 0.05). 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 L R LR Control T3 Mean differences in the number of white bars on juvenile A. ocellaris after control and T3 treatment of larvae. L=Left side, R=Right side. N = 25 fish from each group. Control and treatment groups were significantly different (p < 0.05). DISCUSSION AND FUTURE RESEARCH Immersion of larval Amphiprion ocellaris in T3 induced significant changes in the meristic indices for this species. Subsequent replication of this experiment showed similar but varying changes in the patterning of the white bars, indicating a genetic variability in the response of these fish to exogenous T3. These results indicate a significant role for T3 in larval common clownfish pigmentation and development. Future research will include replication of this experiment with other parentals of the same species, higher and lower concentrations of T3, and replications using T4. Iso-molar concentrations of tyrosine and iodine, the main components of thyroid hormones, will be tested to quantify their contributions to larval clownfish development and pigmentation. Finally, the endogenous larval thyroid hormones will be blocked at the receptor sites with thiourea to quantify the effects of subnormal thyroid hormone levels on metamorphosis. ACKNOWLEDGEMENTS The authors thank Deborah Jung for assistance with the photographs. Bulletin de l’Institut océanographique, Monaco, n° spécial 20, fascicule 1 (2001) REFERENCES BROWN, C.L., and B.G. KIM. 1995. - Combined application of cortisol and triiodothyronine in the culture of larval marine finfish-. Aquaculture 135: 79-86. REDDY, P.K., and T.J. LAM. 1992. - Effect of thyroid hormones on morphogenesis and growth of larvae and fry of telescopic-eye black goldfish, Carassius auratus-. Aquaculture 107 (4): 383-394. Bulletin de l’Institut océanographique, Monaco, n° spécial 20, fascicule 1 (2001) Bulletin de l’Institut océanographique, Monaco, n° spécial 20, fascicule 1 (2001)