FISHERIES SCIENCE 2002; 68: 282–289

INTRODUCTION

A considerable body of information has been com-piled on the role of thyroid hormones in the earlydevelopment of animals. Thyroid hormones havebeen reported to enhance growth and differentia-tion in fishes, and to reduce mortality during the early phase of fish life.1–4 The important roles ofhormones in pigmentation and habitat transferduring metamorphosis have been demonstrated insalmonids,5 black seabream,6 and red seabream.7,8

The coral trout Plectropomus leopardus experi-ences two transformations during its early life,9

similar to Hexagrammos agrammus.10 The firsttransformation occurs at approximately 11–12 mmin standard length (SL), when larvae transform tojuveniles by completing most of their fin formation

and the ossification of vertebrae. The second transformation is at approximately 20 mm SL when resorption of the dorsal and pelvic fin spinestakes place. Settlement, pigmentation, and a surge of thyroxine also occur during this secondmetamorphosis.9

To confirm the contribution of thyroid hormoneto the second metamorphosis of this species, juve-niles between the first and second transformationswere treated with thyroxine (T4) and thiourea (TU;an inhibitor of thyroid hormone synthesis). Mor-phological changes were described as indicators ofthe second metamorphosis.

MATERIALS AND METHODS

Preparation of treatment media

A stock solution of T4 was prepared by dissolving100 mg of L-thyroxine sodium salt (Nacalai Tesque,Kyoto, Japan) in 20 mL of absolute ethanol with afew drops of 1 N NaOH to increase solubility, andkept at 4∞C. Because TU (Nacalai Tesque) dissolves

Original Article

Effects of thyroxine and thiourea on the metamorphosisof coral trout grouper Plectropomus leopardus

Dody Dharmawan TRIJUNO,1a Kenzo YOSEDA,2 Jun HIROKAWA,2 Masatomo TAGAWA1* AND Masaru TANAKA1

1Graduate School of Agriculture, Kyoto University, Kitashirakawa, Kyoto 606-8502 and 2Japan Sea Farming Association, Yaeyama Station, Ishigaki, Okinawa 907-0451, Japan

ABSTRACT: The contribution of the thyroid hormone to the metamorphosis of the coral trout Plectropomus leopardus was examined. Juveniles, aged 35 days, were exposed to thyroxine (T4,0.1 p.p.m) and thiourea (TU; inhibitor of thyroid hormone synthesis, 30 p.p.m) for 13 days. All the fishin the T4-treated group had completed metamorphosis (second metamorphosis, pigmentation andresorption of fin spines) within three days after the beginning of the experiment (day 3), whereas ittook 13 days for this to occur in the control fish. Moreover, the fish in the TU group had not com-pleted metamorphosis even by day 13. Settling behavior was significantly stimulated by T4 treat-ment, as was the resorption of the dorsal and pelvic fin spines, the reduction of serration on thespines, and the transition of body color into an opaque reddish hue. Fish in the TU group had longerdorsal and pelvic spines, retarded pigmentation and an abnormal black coloration. These resultssuggest that thyroid hormone accelerates most changes during the second metamorphosis in thecoral trout.

KEY WORDS: fin spines, grouper, metamorphosis, pigmentation, thiourea, thyroxine.

*Corresponding author: Tel: 81-75-753-6221. Fax: 81-75-753-6229. Email: tagawa@kais.kyoto-u.ac.jp

aPresent address: Department of Fisheries, Faculty of MarineSciences and Fisheries, Hasanuddin University, Jalan PerintisKamerdekaan Km 10, Makassar, Indonesia 90245.

Received 18 May 2001. Accepted 6 September 2001.

Effects of thyroxine on grouper metamorphosis 283

days 3, 6, 9, and 13. The experiment was termi-nated on day 13, corresponding with the completepigmentation of fish in the control group. In addi-tion, on days 0, 3 and 13, three fish from each tankwere anesthetized, fixed in Bouin’s solution, andexamined histologically. Three more fish from eachtank were immediately frozen at –80∞C for T4analysis.11,12

Because pigmentation is a good measure ofmetamorphosis in this species, as shown in arelated species,13 the pigmentation index (PI) wasdetermined according the following stages: A,chromatophores are present only on the dorsalsurface of the head; B, a small number of chro-matophores appear on the anterior dorsal part of the body close to the head; C, many chro-matophores are found on the dorsal side andaround the top of the abdomen; D, chro-matophores cover 50–75% of the body surface; andE, completion of pigmentation (covering most ofthe body). More than 20 fish were observed andclassified before fixation. The numbers of fish that always stayed along the bottom and swamupward only to take prey were counted in thetanks, and the ratio was used as an indicator of settling behavior.

easily in seawater, TU crystals were added directlyto 1 cup of seawater and poured into the experi-mental tanks.

Rearing experiment

Juveniles, aged 35 days [SL 16.71 ± 0.19 mm (mean± SD); N = 27], previously reared in a mass cultureby feeding with rotifer, Artemia nauplii, and anartificial diet were used for the experiment. Thirtyfish were randomly selected and stocked in a 25-L black, circular, polycarbonate tank with mildaeration. Using a tank for each group, the ex-perimental groups comprised the control group,the T4 (0.1 p.p.m) group and the TU (30 p.p.m)group. One-fifth of the rearing water was changeddaily after cleaning the bottom. Water temper-ature and dissolved oxygen were 28.6 ± 0.1∞C and4.5 ± 0.1 p.p.m. (mean ± SD), respectively. Survivalrates for all groups were nearly 100% during theexperiment.

At least five fish from each tank were anes-thetized by adding several drops of 1% MS222 solution, and were then fixed in 10% formalin onday 0 (day experiment was commenced), and

Per

cent

age

Fig. 1 Distribution of pigmentation indices (PI) on days 0, 3, 6, 9, and 13 of the experiment, which are categorizedfrom stages A–E. T4, thyroxine; TU, thiourea.

284 FISHERIES SCIENCE DD Trijuno et al.

Statistical analysis

The effect of treatments on total and standardlength (TL, SL), dorsal and pelvic fin spine lengths(DSL, PSL), pigmentation index (PI), serrationreduction on the dorsal spine (non-serrated spine, NS), and settlement (ST) were tested for significant differences by A N OVA followed by theTukey HSD multiple means comparison test usingthe computer program S P S S (1998) (SPSS Inc.Chicago, IL, USA). The percentage data were trans-formed to arcsin-square root values before testingby ANOVA.14

RESULTS

As shown in Fig. 1, all fish in the T4 group were syn-chronized and fully pigmented (stage E) on day 3,whereas only 17% in the control group and 0% in the TU group had complete pigmentation. Byday 13, all fish in the control group had completepigmentation, whereas 28% of the fish in the TUgroup were fully pigmented (Fig. 1).

As shown typically in Fig. 2, fish in the T4 groupon day 9 had an opaque body, and their color was a bright reddish-orange with dark spots and whitish horizontal stripes when alive; a similar color to that of the metamorphosed fish.Thiourea-treated fish had a transparent body witha dark color on the anterior dorsal and lateral fins,and fish in the control group were midwaybetween both treatments. On day 13 (data notshown), the body coloration of fish in the controland T4 groups was similar; that is, a reddish-orange color with dark spots. Some fish in the TUgroup had complete pigmentation with an abnor-mal black color while others were still widely trans-parent with some black coloration in some areas.No fish in the TU group showed any reddish ororange coloration throughout the experimentalperiod.

Detailed observations of the melanophores tookplace on day 3 (Fig. 3). Some melanophores werestellate in shape while others were similar to theblack dots seen in the control group (C3). In the TUgroup (TU3), most melanophores were stellate,whereas in the T4 group (T4 3) most of themelanophores were similar to black dots. On day13, most of the melanophores in the TU group werelarge and stellate in shape (TU13).

Histological observations on day 3 revealed anincrease in the epidermal thickness of the dorsalskin of the T4 group compared with that of thecontrol and TU groups (Fig. 4). In the T4 group,melanophores were located over and below the

dermal layer, whereas they were present onlyunder the dermal layer in the control and the TUgroup.

The occurrence of non-serrated dorsal andpelvic fin spines (NS) increased significantly (P <0.01) in the T4 group compared with the controland TU groups on day 3 (Fig. 5). The differenceamong groups became smaller by day 13, but was still significantly higher in the T4 group. Settlement behavior (ST) was also significantly stimulated in the T4 group (Fig. 5). All fish in the T4 group completed settlement by day 3, whereasno settled fish were observed in the TU group by day 3. In the control group, only 1% of the fish had settled by day 3; however, by day 13, all fish in the control group had finished settlement,but significantly fewer fish had settled in the TUgroup.

There were no significant differences in the TLand SL among all groups on day 3 (Fig. 6); but SLwas significantly shorter in the T4 group on day 13.

Fig. 2 Pigmentation pattern of representative fish onday 9. Photographs were taken while fish were anes-thetized. C, Control fish [standard length (SL) 20.78 mm,pigmentation index (PI) = DJ; TU, thiourea-treated fish (SL 21.91 mm, PI = C); T4, thyroxine-treated fish (SL 19.46 mm, PI = E).

Effects of thyroxine on grouper metamorphosis 285

Supplementation of T4 into the rearing wateraccelerated the resorption of the dorsal and pelvicfin spines. Conversely, TU retarded the resorp-tion of both spines; these spines were calcified judging by alizarin red staining.9 A similar effect onlarval fin resorption has been clearly observed in Epinephelus coioides13 and the Japanese flounderParalichthys olivaceus.15 In rats, Mundy and col-leagues have shown a direct increase in osteoclas-tic bone resorption by T4 and 3,5,3¢-triiodo-L-thyronine (T3) in vitro,16 which could be the reasonfor the enhancement of larval fin spine resorptionby T4 treatment in the coral grouper. A stimulatingeffect by T3 in bone formation and resorption inrainbow trout Oncorhynchus mykiss has also beenreported.17

In the TU group, changes in morphologicalcharacteristics (fin rays and color) were signifi-cantly inhibited, but settling behavior was inhib-ited to a lesser degree. Similar differential effects of thyroid hormone have been reported in theJapanese flounder; that is, TU did not completely

The standard length of dorsal and pelvic spines(DSL and PSL) were significantly (P < 0.01) shorterin T4-treated fish on both day 3 and day 13 (Fig. 7).The DSL and PSL were significantly greater in theTU-treated fish on day 13.

Whole body T4 levels were highest (ª200 ng/g) inthe T4 group (P < 0.01), and lowest in the TU group(ª2 ng/g) (Fig. 8). Thyroxine levels in the controlfish were approximately 15 ng/g and remainedconstant during the experiment.

DISCUSSION

The involvement of thyroid hormones in fish metamorphosis has been demonstrated in manyspecies.3,10,13,15 In the present study, T4 had anaccelerating effect on metamorphosis in the coraltrout, whereas TU clearly demonstrated a retardingeffect. Thyroxine seemed to synchronize the timingof the metamorphosis because all fish underwentmetamorphosis at the same time.

Fig. 3 Melanophore condition of 10% formalin-fixed fish. C3, control fish on day 3; T4 3, thyroxine-treated fish on day 3; TU3, thiourea-treated fish on day 3; TU13, thiourea-treated fish on day 13. Arrows indicate melanophores. Bars = 100 mm.

286 FISHERIES SCIENCE DD Trijuno et al.

inhibit right eye migration but effectively retardedsettling behavior and the resorption of elongatedfin rays.15 There are two possible explanations for this effect. First, although TU suppressed theendogenous level of T4, a small amount of T4might still be sufficient and necessary to stimulatesettling behavior at a slow rate. Second, settlingbehavior does not require T4 at all. This seems tosuggest differences in the extent that the thyroidhormone is required among different tissues andfor behavior.

The effects of T4 treatment on pigmentation areknown in several fishes; for example, silveringduring smoltification in salmonid fishes,5 earlierappearances of melanophores on the black stripein red seabream Pagrus major,7 and body colorchanges in the spottybelly greenling Hexagrammosagrammus.10 In the present study, the treatment ofT4 clearly accelerated pigmentation. Thyroxine-treated fish were of an opaque reddish color,whereas TU-treated fish were transparent and par-tially covered by a black color. During the meta-morphosis of the Japanese flounder, juvenile-type(smaller size) melanophores appear in addition tothe larval type (bigger size).18 In the present study,the ‘appearance’ of melanophores after fixationwas also affected by the long-term treatment of thethyroid hormone; that is, dot-like appearances inthe T4-treated fish and stellate appearances in theTU-treated fish. The results of the experiment sug-gests the contribution of the thyroidal system inthe function and/or formation of melanophores in the coral trout. Chromatophores other thanmelanophores were not examined in the study, butdetailed observation of erythrophores is necessaryto understand the mechanisms of T4-dependentpigmentation during the second metamorphosisof this species.

In addition to the effect on melanophores,hypertrophy of the epidermis was observed in theT4-treated group in the present study. Increasedthickness of the epidermis by thyroid hormoneshas been found previously in platyfish19 and intilapia Sarotherodon niloticus.20 Thickness of thedermis was also increased by T4 treatment insalmonid fish.21 These findings are in accordancewith the results of the present experiment.

The present study has clearly demonstrated that T4 stimulates metamorphosis in coral trout,and that TU retards the process. This suggests that the thyroidal system plays a major role in the regulation of metamorphosis in the coral trout.

ACKNOWLEDGMENTS

We thank all the staff at Japan Sea Farming Association (JASFA), Yaeyama Station. This studywas done under a cooperative program between JASFA and Kyoto University and was partly sup-ported by a scholarship awarded to DDT from the Ministry of Education, Science, Sports and Cul-ture, Japan, and partly by Grants-in-Aid from theMinistry of Education, Science and Culture, Japanand from the Ministry of Agriculture, Forestry andFisheries, Japan, which were each awarded to MT and MT.

Fig. 4 Skin sagittal sections of fish sampled on day 3. C,Control; TU, thiourea-treated fish. T4, thyroxine-treatedfish; Ep, epidermis; Mc, mucus cell; D, dermis. Bars =20 mm.

Effects of thyroxine on grouper metamorphosis 287

Per

cent

age

** **

Fig. 5 Percentage of fish with (�) non-serrated dorsal spines (NS) and ( ) those that settled (ST) on days 0, 3 and 13of the experiment. More than 20 fish were examined from each tank for NS. Vertical bars indicate standard errors ofthe means; n = 3 tanks; T4, thyroxine; TU, thiourea. Significantly different from control at *P < 0.01 and **P < 0.05.

Fig. 6 (�) Total length and ( ) standard length on days 0, 3 and 13 of the experiment. Vertical bars indicate standarderrors of the means; n > 20 fish; T4, thyroxine; TU, thiourea. *Significantly different from control (P < 0.01).

288 FISHERIES SCIENCE DD Trijuno et al.

Fig. 7 Standard length of the dorsal (DSL; �) and pelvic ( PSL; ) spines on days 0, 3 and 13 of the experiment. Vertical bars indicate standard errors of the means; n = 15 fish; T4, thyroxine; TU, thiourea. *Significantly different from control (P < 0.01).

T4

conc

entr

atio

n (n

g/g

body

wei

ght)

Fig. 8 Whole body T4 (thyroxine) levels on days 0, 3 and 13 of the experiment. Vertical bars indicate standard errorsof the means; n = 3 pools of three fish; TU, thiourea. *Significantly different from control (P < 0.01).

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13. de Jesus EG, Toledo JD, Simpas MS. Thyroid hormonespromote early metamorphosis in grouper (Epinepheluscoioides) larvae. Gen. Comp. Endocrinol. 1998; 112: 10–16.

14. Zar JH. Biostatistical Analysis. Prentice Hall International,Upper Side River, NJ. 1999.

15. Miwa S, Inui Y. Effects of various doses of thyroxine and triiodothyronine on the metamorphosis of flounder (Paralichthys olivaceus). Gen. Comp. Endocrinol. 1987; 67: 356–363.

16. Mundy GR, Shapiro JL, Bandelin JG, Canalis EM, Raisz LG.Direct stimulation of bone resorption by thyroid hormones.J. Clin. Invest. 1976; 58: 529–634.

17. Takagi Y, Hirano J, Tanabe H, Yamada J. Stilmulation ofskeletal growth by thyroid hormone administrations in therainbow trout, Oncorhynchus mykiss. J. Exp. Zool. 1994; 268:229–238.

18. Seikai T. Process of pigment cell differentiation in skin on the left and right sides of the Japanese flounder,Paralichthys olivaceus, during metamorphosis. Jpn. J.Ichthyol. 1992; 39: 85–92.

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