changes in growth performance and proximate composition in japanese flounder during metamorphosis

Changes in growth performance and proximate

composition in Japanese flounder during metamorphosis

M. A. R. HOSSAIN*†, M. TAGAWA*, R. MASUDA‡ AND

M. TANAKA*

*Laboratory of Marine Stock-enhancement Biology, Division of Applied Bioscience,Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan and‡Fisheries Research Station, Kyoto University, Naga-Hama, Maizuru, Kyoto,

625-0086, Japan

(Received 15 January 2002, Accepted 22 August 2003)

Morphological changes are described in Japanese flounder Paralichthys olivaceus larvae and

juveniles with emphasis on growth during the period of metamorphosis. Ontogenetic changes in

condition factor and lipid, glycogen and protein levels were also analysed to determine the

utilization of stored energy. Fish grew from 6�6 to 20�3mm LT (1�15 to 84�4mg in mass, M)

during the period from 11 to 40 days after hatching (DAH) at 19�3� C. Per cent specific growthrate per day (% G day�1) for wet mass was lowest during the metamorphic phase (21–30DAH)

compared to pre- (11–20DAH) and post-metamorphic (31–40DAH) phases. When LT and M

were expressed as M¼ a LbT, the b value was highest during the pre-metamorphic phase and

lowest during the metamorphic phase. These findings indicate that the developmental changes

that occur during metamorphosis of Japanese flounder are closely related to the growth pattern.

Moisture, lipid and glycogen contents were also at the lowest level during metamorphosis

compared to pre- and post-metamorphosis, which suggest that Japanese flounder use their

energy reserves to accomplish metamorphosis due to an apparent decline in feeding during this

period. # 2003 The Fisheries Society of the British Isles

Key words: growth retardation; metamorphosis; Paralichthys olivaceus; settlement; stored

energy.

INTRODUCTION

The Japanese flounder Paralichthys olivaceus (Temminck & Schlegel) is one ofthe most important and the highest-priced fishes in the coastal fisheries ofJapan, with an annual catches of 6000 to 8000 t (Tanaka et al., 1989; Fujii &Noguchi, 1996). Because they are easy to handle, aquaculture of juveniles of thisspecies is flourishing in Japan. Now Japanese flounder is the most importanttarget species for stock enhancement of marine fishes with an annual release of25 million hatchery-reared juveniles.Like other pleuronectiforms, Japanese flounder larvae go through a meta-

morphosis coincident with the shift from pelagic to benthic life and from

†Author to whom correspondence should be addressed at present address: Department of Fish Biology

and Genetics, Faculty of Fisheries, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

Tel.: þ880 91 54963; fax: þ880 91 55810; email: [email protected]

Journal of Fish Biology (2003) 63, 1283–1294

doi:10.1046/j.1095-8649.2003.00251.x,availableonlineathttp://www.blackwell-synergy.com

1283# 2003TheFisheries Society of theBritish Isles

Administrator

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symmetric larvae to asymmetric juveniles (Tanaka et al., 1989). Youson (1988)characterized this metamorphosis (‘first metamorphosis’) as a dramatic andabrupt change in form that is triggered by internal and environmental cues.Morphological changes during metamorphosis in this species are described indetail, and the transition forms staged, by Minami (1982). The metamorphosisis known to be triggered by thyroid hormones (Inui et al., 1989; Tagawa et al.,1990) and the timing fluctuates depending on environmental factors such aswater temperature (Seikai et al., 1986).Fukuhara (1986) described the morphological and functional development

including eye migration and swimming speed of Japanese flounder in early lifestages. The effect of temperature on the duration of different stages of meta-morphosis of the species was described by Seikai et al. (1986). The activity ofthyroxine at two different temperatures (Tanangonan et al., 1989) and differentfatty acids in the proximate composition (Furuita et al., 1998) of metamorphos-ing Japanese flounder have also been investigated. Tanaka et al. (1996)described the morphology of the developing digestive system in relation tometamorphosis and settlement. Although the early life history of Japaneseflounder has been extensively studied (Tanaka et al., 1998), day to day growth,growth retardation (if any) and feeding of this species during different phases ofmetamorphosis is still not fully understood. The first aim of the present studywas to describe, growth performance before, during and after metamorphosis.The second aim was to analyse developmental changes in body composition(lipid, glycogen and protein content) to elucidate their role in metamorphosis.

MATERIALS AND METHODS

FISH AND REARING CONDITIONS

Japanese flounder eggs were provided by the Japan Sea Farming Association(JASFA), Miyazu Station, Miyazu, Kyoto Prefecture, Japan. Eggs were hatched on5 May 2000 and larvae were reared at the Fisheries Research Station (FRS) of KyotoUniversity, Maizuru, Kyoto Prefecture, Japan, during May to June, 2000. Eggs wereincubated in a 500 l polycarbonate tank with running sea water. Water in the tank wasaerated at a rate of 50–100mlmin�1. Larvae were fed L-type rotifers Brachionus plicatalis(O.F. Muller) (supplied by JASFA Miyazu Station and cultured with Chlorella sp.) for21 days, from 3 days after hatching (DAH). Artemia nauplii (Miyako Kagaku Co. Ltd.,Tokyo, Japan) were fed to larvae from 10–35DAH. At the end of the Artemia feeding,26DAH, fish were gradually switched to pellet food until the juveniles reached 5 cm totallength (LT). Both rotifers and Artemia nauplii were enriched with docosahexaenoic acid(DHA, Harima Kagaku Co. Ltd, Tokyo, Japan) and grown at an incubation density of200–300 individualsml�1 and 100 individualsml�1, respectively. They were disinfectedusing the anti-bacterial agent, Erbaju (10% beads, Ueno Fine Chemical Industry Ltd,Osaka, Japan) before being fed to Japanese flounder larvae. Temperature in therearing tank was maintained at 19�3� 0�3� C (mean� S.E.). Controlled illuminationwith fluorescent lamps gave a 12L : 12D photoperiod (light on 0830 hours).

SAMPLING AND PRESERVATION

Fish were sampled every day between 11–40DAH (total sampling period 30 days). Allthe sampled fish were first anaesthetized using 200 ppm benzocaine (MS222). A few ofthese fish were examined under a microscope and the food items (rotifer or Artemia) in

1284 M. A . R . HOSSAIN ET AL .

# 2003TheFisheries Society of the British Isles, Journal of FishBiology 2003, 63, 1283–1294

the stomach were counted. Then half of the remaining sampled fish were frozen at�80� C and the other half was preserved in 10% buffered formalin solution.

DEVELOPMENTAL STAGES

Formalin preserved sample were carefully separated into developmental stages as firstdescribed by Minami (1982) and later modified by Seikai et al. (1986) and Tanangonanet al. (1989). These authors classified the development of Japanese flounder larvae intothree main phases; pre-metamorphosis, metamorphosis and post-metamorphosis with tensub-stages (Table I). All the stages are defined based on the morphological and behav-ioral characteristics of larvae.

MORPHOMETRIC MEASUREMENTS AND PROXIMATECOMPOSITION ANALYSES

Alizarin staining was done to identify fin rays, eye migration and skeletal development.The formalin preserved samples were stained based on Sire et al. (1997) with a fewmodifications. First, the samples were rehydrated in water and depigmented forc. 20min in 0�5% KOH containing 3% H2O2 followed by rinsing in distilled water toremove H2O2. The fish samples were then placed in a mixture of 1% KOH and glycerol(1v/1v) for 2 h for partial clearing. Then the samples were stained using alizarin reddissolved in 0�5% KOH. After staining the samples were cleared again in the samemixture (KOH and glycerol) for 1 h and finally stored in pure glycerol. Body dimensionsmeasured were LT (cm), standard length (LS, cm), mass (M, g) and body depth (BD,mm). Body length and depth measurements were made using a microscope with a profileprojector. Average daily growth was calculated in each phase for LT as 0�1 (LTend –LTstart) and for mass as 0�1 (Mend –Mstart). The specific growth rate (G, % day�1) wascalculated as G¼ 100 g, where g¼ (lnX2� lnX1) (t2� t1)

�1 and X1 and X2 are LT, LS, BDand M at times (days) t1 and t2. (Busacker et al., 1990).The relationship between M (g) and LT (cm) was calculated from M¼ a Lb

T, wherea and b are constants. When b¼ 3, growth is isometric (Ricker, 1979). The nutritionalcondition of the fish was estimated using the condition factor K (Bolger & Connolly,1989), calculated as: K ¼ 100ML�3

T , where LT is in cm.Moisture, protein and lipid content were estimated according to Fonds et al. (1995).

Glycogen content was analysed following the method detailed by Montgomery (1957).

STATISTICAL ANALYSIS

Moisture, protein, lipid and glycogen content was expressed as per cent body mass andarcsine transformed. Bartlett’s test revealed homogeneous variances for all the statisticalcomparisons (Sokal & Rohlf, 1981). Growth variables and proximate composition oflarvae at the three stages were analysed by ANOVA (a¼ 0�05).

RESULTS

METAMORPHIC PHASES

Larvae at 11 and 13DAH (average LT¼ 6�6 and 7�8mm, respectively) hadsymmetrically positioned eyes and three elongated dorsal fin rays. They wereidentified as stage C (Fig. 1). Some larvae at 12DAH, however, had D stagecharacteristics with five dorsal fin rays and six caudal fin rays (Fig. 1). At15DAH (average LT¼ 8�8mm) the body depth was expanded, notochord flex-ion was observed, and thus larvae were identified as stage E. Some of the larvae,however, were found to be at stage D even after 15DAH.

JAPANESE FLOUNDER METAMORPHOSIS 1285

# 2003TheFisheries Society of theBritish Isles, Journal of FishBiology 2003, 63, 1283–1294

TABLEI.

Morphometricandbehaviouralcharacteristics

ofmetamorphosingJapaneseflounder

larvaein

differentstages

Development

Stage

Characteristics

Pre-m

etamorphosis

AEyes

symmetrical.Yolk

totallyabsorbed

butoilglobule

sometim

espresent.

Openingofmouth.Startsexogenousfeeding.Pelagic

larvae.

BEyes

symmetrical.Budofelongateddorsalfinrays.Gutexpanded.Pelagic

larvae.

CEyes

symmetrical.Threeelongatedanteriordorsalfinrays.Budofcaudalfinrays.Pelagic

larvae.

DEyes

symmetrical.Beginningofbodycompression.Fiveelongateddorsalfinrays.Six

caudalfinrays.Pelagic.

ENotochord

flexion45�upward.Bodydepth

increased.Beginningofeyemigration.Pelagic

larvae.

Earlymetamorphosis

FSix

elongateddorsalfinrays.Proliferationofdorsalandanalfinrays.Slightlydorsal

movem

entofrighteyebutstillrighteyeunobservable

from

leftside.

Definiteasymmetry

dueto

eyemigration.Notochord

stillextendingposteriorto

hypurals.Hypuralsandcaudal

finraysdeveloping.Pelagic

larvae.

Mid-m

etamorphosis

GRighteyeobservable

from

leftside.

Dorsalandanalfinrayswelldeveloped.Pelagic

larvae.

Late

metamorphosis

HNearlyhalfoftherighteyeobservable

from

leftside.

Righteyeatdorsalmid

lineordorsaledge.

Bodydepth

increased.Pelagic

withperiodicalsettlingbehaviour(pseudo-settlem

ent).Metamorphic

clim

ax.

Post-m

etamorphosis

I 1Righteyecompletely

ontheleftside.

Elongateddorsalfinraysabsorbed.Settled

orbenthic

juveniles.

I 2Settled

orbenthic

juveniles

for5days.

After

Minami(1982),Seikaiet

al.(1986)andTanangonanet

al.(1989).



The first sign of eye migration was observed at 18DAH and the early stage ofmetamorphosis (stage F) started from 19 to 23DAH (LT¼ 10�1–12�1mm). At22DAH (13�3mm) the right eye reached the dorsal ridge and was observablefrom the left side (mid-metamorphosis, stage G). Well developed dorsal andanal fins were formed at this stage.At 25DAH, larvae occasionally settled on the bottom of the tank for a short

period (late metamorphosis, stage H). Feeding activity at this stage was tem-porarily decreased. At 28DAH fish showed periodical settling behaviour, withtheir right eye completely migrated and the dorsal fin fold resorbed (post-metamorphosis, stage I). By 33DAH, all fish had completely metamorphosedinto juveniles measuring 13�4–16�4mm LT. They ceased pelagic life and settledon the tank bottom or tank wall.For further comparisons in this paper, fish on 11–20, 21–30 and 31–40DAH

were defined as pre-metamorphic, metamorphic and post-metamorphic, respect-ively.

GROWTH PERFORMANCE

Over the whole experimental period the constant b from the mass–lengthrelationship was 3�56 (Fig. 2). The value of b was highest (4�85) in the pre-metamorphic, lowest (2�96) in the metamorphosis and intermediate (3�25) dur-ing the post-metamorphic phases. The condition factor increased rapidly duringthe pre-metamorphic phase, then stabilized with some fluctuation during the

10 15 20 25 30 35 40

I

H

G

F

E

D

C

6.5 10.5 14.5 18.5 22.5

Start End

Days after hatching

Dev

elop

men

tal s

tage

s

LT (mm)

FIG. 1. The duration of different developmental stages of Japanese flounder larvae during the experimental

period (between 11 and 40 days after hatching).



metamorphic phase. Average K in fish over the whole experimental period was0�88. The value of K was lowest (0�68) in the metamorphosis phase, highest(1�00) in the pre-metamorphic phase and intermediate (0�94) during the post-metamorphic phase (Table II).Average daily growth in pre-metamorphosis, metamorphosis and post-

metamorphosis was 0�42, 0�34 and 0�72mmday�1 (LT) and 1�07, 1�55 and 5�14mgday�1 (M). The specific growth rate for wet mass was lowest (P< 0�05) duringmetamorphosis followed by post-metamorphosis and pre-metamorphosis (Fig. 3).The specific growth rate measured by LT, LS and BD depth were highest in

pre-metamorphic phase, then decreased towards metamorphosis and againincreased after the metamorphosis (Fig. 4).

FISH BODY COMPOSITION

Moisture, lipid and glycogen levels decreased during metamorphosis (DAH21–30) and then recovered to higher level after metamorphosis (Table II andFigs 5 and 6). Protein level did not appear to decline during the metamorphicphase (Table II and Fig. 5).

DISCUSSION

GROWTH DECLINE DURING METAMORPHOSIS

Growth rate of Japanese flounder declined during metamorphosis (Figs 3 and4). The LT at post-metamorphosis found in this experiment is in agreement withSeikai et al. (1986). The average daily length increment, however, was better in

0.500.00

0.02

0.04

0.06

0.08

0.10

1.00 1.50 2.00 2.50

10 15 20 25 30 35 40

Days after hatching

Mas

s (g

)

LT (cm)

FIG. 2. The length and wet mass relationship of metamorphosing Japanese flounder larvae. Error bars

are S.E. The curve was fitted by M¼ 0�007 L3�56T (r2¼ 0�98).



TABLEII.Mean�

S.E.conditionfactorandproxim

ate

compositionofJapaneseflounder

larvaein

threephasesofmetamorphosis.

Values

withdifferentsuperscriptlettersweresignificantlydifferent(P

<0� 05)amongthephases

DAH

KMoisture

(%)

Dry

mass

(%)

Lipid

(%wet

mass)

Lipid

(%dry

mass)

Protein

(%wet

mass)

Protein

(%dry

mass)

Glycogen

(%dry

mass)

Glycogen

(%wet

mass)

11–20

1� 00�0� 03c

81� 73�0� 48c

18� 27�0� 48a

2� 78�0� 05c

15� 38�0� 63c

11� 54�0� 17ab

63� 43�1� 40b

0� 82�0� 04d

0� 15�0� 01c

21–30

0� 68�0� 06a

78� 83�0� 20a

21� 17�0� 20c

1� 62�0� 13a

7� 71�0� 68a

12� 78�0� 20c

60� 41�1� 11a

0� 31�0� 04a

0� 06�0� 01a

31–40

0� 94�0� 07bc81� 26�0� 39bc18� 74�0� 39a

2� 15�0� 20b

11� 70�1� 20b

11� 28�0� 21a

60� 24�0� 81a

0� 49�0� 06b

0� 09�0� 01b

11–40

0� 88�0� 06b

80� 60�0� 32b

19� 40�0� 32b

2� 19�0� 12b

11� 59�0� 79b

11� 87�0� 17b

61� 36�0� 72ab

0� 54�0� 05c

0� 10�0� 01b



0

10

20

30

40

11–20 21–30 31–40

G (

% d

ay–1

)

Days after hatching

c

a

b

FIG. 3. Mean� S.E. specific growth rates for wet mass at three different phases. Different letters indicate

significantly different values (P< 0�05).

0.0

2.5

5.0

10 20 30 40

0.0

2.5

5.0

10 20 30 40

0.0

2.5

5.0

10 20 30 40

Day after hatching

G (

% d

ay–1

)

(a)

(b)

(c)

FIG. 4. Daily specific growth rates of larval Japanese flounder over the experimental period based on (a)

total length, (b) standard length and (c) body depth.



the present experiment (0�72mm) than what was reported (0�59mm) by Seikaiet al. (1986) during post-metamorphosis at the same rearing temperature (19� C).A decline in growth in metamorphosing Pleuronectiformes has been reported

in several species. Laroche (1982) demonstrated that wild English soleParophrys vetulus Girard do not grow at all during metamorphosis. Similarphenomena were reported in marbled sole Limanda yokohamae (Gunther)(Fukuhara, 1988) and brown sole Pleuronectes herzensteini (Jordan & Snyder)(Aritaki et al., 1996) in hatchery reared fishes. The growth of winter flounderPleuronectes americanus (Walbaum) slows down but does not cease during

0

20

40

60

80

10 15 20 25 30 35 40

Per

cen

t of

dry

bod

y m

ass

Days after hatching

FIG. 5. Mean� S.E. protein (*) and lipid (*) as a percentage of dry body mass of larval Japanese

flounder during the experimental period.

0.0

0.4

0.8

1.2

10 15 20 25 30 35 40

Per

cen

t of

dry

bod

y m

ass

Days after hatching

FIG. 6. Mean� S.E. glycogen level (as a percentage of dry body mass) of larval Japanese flounder during

the experimental period.



metamorphosis (Bertram et al., 1997), similar to the present results for Japaneseflounder.Japanese flounder in the present work showed less feeding activity during

metamorphosis (unpubl. obs.). This is consistent with observations by Tanakaet al. (1996) that newly settled Japanese flounder juveniles in the wild tend tohave empty stomachs. Keefe & Able (1993) also reported that hatchery rearedsummer flounder Paralichthys dentatus (L.) had empty stomachs in the meta-morphosis phase. In the present study, Japanese flounder showed a particularlysubstantial decline in growth for a few specific days within the overall meta-morphosis phase (DAH 21–30) probably due to reduced feeding.

PROXIMATE COMPOSITION AND METAMORPHOSIS

Proximate composition in metamorphosing Japanese flounder in the presentstudy was close to that reported in larvae (Furuita et al., 1998) and juveniles(Kikuchi et al., 1994; Kim & Lall, 2000). The decrease in lipid and glycogencontent during metamorphosis, suggested that energy sources for metamorphosiscame from these sources of reserved energy. Fluctuations of K also indicated theunstable nutritional status of the larvae at this stage. Since metamorphosinglarvae eat less but require more energy (than normal condition) to accomplishdrastic morphological and physiological changes, they use much of their lipid andglycogen reserves. Similar results have been reported in bonefish Albula sp.larvae, where 80% of the energy requirements for the metamorphosis fromleptocephali was provided by the breakdown of lipids (Padron et al., 1996).Lowered lipid levels in metamorphosing larvae are also reported in cod Gadusmorhua L. (Fraser et al., 1988) and striped bass Morone saxatilis (Walbaum)(Chu & Ozkizilcik, 1995).Unlike lipid and glycogen, the protein level did not show a drastic decrease

during metamorphosis. This indicates that structural body musculature is con-served during the energetically challenging metamorphosis period in favour ofusing lipid and glycogen reserves.High mortality may be induced because lack of energy reserves accumulated

during the pre-metamorphic phase that could result in a shortage of storedenergy for use during metamorphosis. More specific identification of the typesof lipid and glycogen are required to fully understand their role in the mechan-ism of metamorphosis.

We thank all at the Fisheries Research Station of Kyoto University. We thank JASFAMiyazu Station for providing Japanese flounder eggs. Comments from two anonymousreviewers and the editor substantially improved the quality of the manuscript. Thisresearch was funded by the Japan Society for the Promotion of Science (JSPS) and theMinistry of Education, Culture and Science (Monbukagakusho).

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changes in growth performance and proximate composition in japanese flounder during metamorphosis

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