048-Clement

Bulletin de l’Institut océanographique, Monaco, n° spécial 20, fascicule 1 (2001)
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
1Fisheries and Illinois Aquaculture Center, Southern Illinois University at
Carbondale, Carbondale, IL 62901, USA
2Reef 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-L-
thyronine (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.
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.
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).
0.0
2.0
4.0
6.0
8.0
10.0
12.0
Control T3
# Dorsal Spines
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Control T3
# Bars
L
L
R
R
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).

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)