Ž .Aquaculture 202 2001 359–370www.elsevier.comrlocateraqua-online

Temperature effects on muscle growth in twož /populations Atlantic and Mediterranean of

sea bass,Dicentrarchus labrax L.

Marıa D. Ayalaa, Octavio Lopez-Alborsa,), Francisco Gila,´ ´Alicia Garcıa-Alcazarb, Emilia Abellanb, Juan A. Alarconc,´ ´ ´ ´

´ c aMarıa C. Alvarez , Gregorio Ramırez-Zarzosa ,´ ´Francisco Morenoa

a Anatomıa y Embriologıa, Fac. Veterinaria, UniÕersidad de Murcia, 30100 Murcia, Spain´ ´b ( )Centro Oceanografico de Murcia I.E.O. , Ctra. Azohıa srn 30860 Pto. de Mazarron, Spain´ ´ ´

c Dept. de Genetica. Fac. de Ciencias. UniÕersidad de Malaga, Malaga, Spain´ ´ ´

Abstract

Ž .Two genetically different populations of sea bass,Dicentrarchus labrax L., Atlantic Atl. andŽ . Ž .Mediterranean Med. , were subjected to the following incubationrcultivation temperaturesT :

Ž15 8Crnatural, 178Crnatural, 15r19 8C, 17r19 8C natural T averaging 158C and raising.gradually . Muscle cellularity was measured at different larval stages for eachT regime. During

the vitelline phase, muscle growth was mainly due to muscle fibre hypertrophy. In Med. larvae,Ž .higher incubationT 17 8C increased the area of white and red fibres at hatching, while in Atl.

larvae there was no significantT effect at this stage. At mouth opening, the area of white fibresŽ .increased at 198C in Atl. larvae P-0.05 , but in Med. larvae it was similar for all temperatures.

Following yolk-sac reabsorption, hypertrophy and hyperplasia increased in both populations. InŽ . Ž .these stages 20–55 days , both parameters were greater at 198C P-0.05 . Metamorphosis

finished earlier at 198C. At this stage, Atl. larvae reared at 198C showed higher value of totalmyotomal area than at naturalT, while in Med. sea bass, larvae reared at 198C showed a lowersize of the myotome than at 178Crnatural. In larvae from both populations reared at naturalT,incubatingT had a positive effect at the end of metamorphosis, thus the total myotomal area werehigher at 178Crnatural than at 158Crnatural. Following metamorphosis, all groups showed a

Ž .rapid growth, but higher at 198C P-0.05 . The results indicate that muscle cellularity wasclearly influenced byT, and that both populations had different levels of response. These

) Corresponding author. Tel.:q34-96-8364694; fax:q34-96-8364147.Ž .E-mail address: albors@fcu.um.es O. Lopez-Albors .´

0044-8486r01r$ - see front matterq2001 Elsevier Science B.V. All rights reserved.Ž .PII: S0044-8486 01 00785-2

( )M.D. Ayala et al.rAquaculture 202 2001 359–370360

differences can be apparently associated with their respective genetic background.q2001 ElsevierScience B.V. All rights reserved.

Keywords: Sea bass; Muscle growth; Temperature; Genetics

1. Introduction

Ž .TemperatureT has a marked effect on muscle growth and developmental rate ofteleosts. Sea bass is a commercially farmed species and the influence ofT on musclegrowth can be of practical interest for aquaculture.T has been shown to influence thenumber and size distributions of the muscle fibres in the teleosts. Nathanailides et al.Ž .1996 investigated theT effect on the growth of juveniles of European sea bass. These

Ž .authors observed that muscle growth was higher at 16–208C naturalT than at 138CŽ .cold T , and that it was mediated through an increase in the number of nuclei andmuscle fibres. However, studies onT effect on the larval development are less advancedthan those on juveniles, and this is the case for sea bass. Such studies may be ofpractical interest in aquaculture since during early life historyT influences future

Ž .growth. Thus, Johnston et al. 1998 incubated eggs of herring,Clupea harengus L., at5, 8 or 12 8C and observed that embryonicT affected the subsequent rate of musclegrowth throughout the larval stages. The effects ofT on muscle cellularity producesintra- and interspecific variations, as has been reported in Atlantic salmon,Salmo salarŽ . Ž .Johnston and McLay, 1997 andC. harengus Johnston et al., 1996 , and have beenassociated to genetic variation andror differences in factors that influence larval growth,such as egg size and quality.

In the present study, different sets of incubatingrcultivating T have been applied toAtl. and Med. sea bass samples to assess the existence of genetic influence on themuscle cellularity of this species. This study is intended to contribute to a betterunderstanding of the relationship among muscle growth,T response and the geographicorigin of the experimental stock.

2. Material and methods

ŽThese experiments were carried out at the Instituto Espanol de Oceanografıa Centro˜ ´.Oceanografico de Murcia, Mazarron , using eggs from spawners of Atlantic and´ ´

Mediterranean origin, which were adapted to captivity. To determine the level of geneticdifferentiation, both broodstocks were screened for two microsatellite loci, named Dla 6

Ž .and Dla 11 Castilho and McAndrew, 1998 . According to the allelic frequenciesobtained, both populations seem to be in Hardy–Weinberg equilibrium for the two loci.To estimate the differentiation level between both populations, the Fst values were

Ž .calculated according to Weir and Cockerham 1984 . For the locus Dla 6 a Fst value of0.010, Ps0.03 was obtained. For the locus Dla 11, Fsts0.0249, Ps0.02. Thesevalues can be interpreted, in terms of population genetics, as the Atl. and Med.populations being different with a probability of 97% and 98%, respectively. Data were

Ž .analysed with the software Genetix 3.3 Belkhir et al., 1998 .

( )M.D. Ayala et al.rAquaculture 202 2001 359–370 361

Eggs from both populations were obtained from the end of January until theŽ .beginning of February 1997 at naturalT (15 8C . Eggs incubation was performed in

Ž 3.cylindricoconical tanks 0.5 m and prelarvae and larvae cultivation was carried out inŽ 3. Ž .cylindrical tanks 1 m . The experimental groups four per population were maintained

under the following incubationrcultivation conditionT ’s: 15 8Crnatural, 15r19 8C, 17Ž .8Crnatural and 17r19 8C Fig. 1 . NaturalT averaged 158C at the beginning of the

experiment and raised gradually. Sampling points were chosen when animals reached

Ž . Ž . Ž .Fig. 1. a,b Incubation and cultivationT regimes of Atlantic sea bass a and Mediterranean sea bass b .

( )M.D. Ayala et al.rAquaculture 202 2001 359–370362

Ž .defined live stages Table 1 , which coincided with different days post-hatchingŽ .depending on the experimental groups Table 1 . Ten animals from each stage and tank

were randomly chosen. Fish were overexposed to the anesthetic with tricaine methane-Ž .sulphonate MS222, Sigma and subsequently fixed in 2.5% glutaraldehyde in buffered

Ž .0.1 M cacodylate pH 7.2–7.4 for 3 h, at 48C. Then, samples were processed forŽ .histological studies according to Wanson and Drochman’s 1968 method. Semi-thin

Ž .sections 1-mm thick were cut transversely to the long body axis, at the point of theanal opening with a Reichert Jung ultramicrotome. Semi-thin sections were stained using

Ž .the technique of Ontell 1974 . Due to the large size of the larvae at 90 days, the caudalhalf of larvae, cross-sectioned at the level of the anal opening, were frozen in2-methylbutane, snap-frozen over liquid nitrogen. Subsequently, sections of 8-mmthickness were obtained in a Reichert Jung cryostat and then stained with haema-toxylinreosin. Muscle growth quantification was carried out from five individuals foreach sampling point and experimental group, by the morphometric analysis of muscleŽ .IMCO 10 System Kontron bildanalyse . The following parameters were measured: totalcross-sectional area of the red and white muscles and total cross-sectional area of themyotome; number of red and white muscle fibres; and the average cross-sectional areaof the red and white muscle fibres. At hatching and mouth opening, all muscle fibreswere measured. From 20 days on, because of the large number of white fibres, theintermediate sector of the myotome thickness was chosen. The counts of fibres werecarried out directly from photographs at an adequate magnification. At 90 days, due to

Žlarge size of the larvae, only the transverse area of white muscle was measured in two.to four larvae per tank . The body length of 10 larvae was measured every 2–10 days in

each tank throughout the whole experiment, while the body weight was obtained every

Table 1Ž .Sampling points: developmental stages hatching, mouth opening and scaling and nutritional events

Ž .Physiological and Sampling points days post-hatchnutritional events Atl. D. labrax Med. D. labrax

ŽHatching end of 0 d. 0 d.a.embryonic stage 0 d. 0 d.

Ž . Ž .Mouth opening 4 d. 17r19 8C 3 d. 17r19 8CŽ . Ž . Ž .end of prelarval stage 4 d. 15r19 8C 4 d. 15r19 8C

Ž . Ž .6 d. 178Crnat. 5 d. 178Crnat.Ž . Ž .6 d. 158Crnat. 6 d. 158Crnat.

Live feeding 20 d. 20 d.Ž .nauplii of ArtemiaBeginning of inert feeding 48 d. 55 d.

Ž ŽEnd of metamorphosis 52 d. 15r19 8C 52 d. 15r19 8CŽ . . .scaling and 17r19 8C and 17r19 8C

Ž Ž .71 d. 158Crnat. 67 d. 178Crnat.. Ž .and 178Crnat. 73 d. 158Crnat.

Ž .Postlarval stage alevins 90 d. 90 d.

d.sdays; nat.snatural.aIncubation time in Atl.D. labrax was 3 days at 178C and 4 days at 158C. Incubation time in Med.D.

labrax was 2.5 days at 178C and 3 days at 158C.

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2–10 days from 40 days until 90 days. The results were statistically analysed by analysisŽ .of variance ANOVA, P-0.05 using the Systat 5.0 Win programme.

3. Results

3.1. Temperature effects on the deÕelopmental rate

The rate of development ofDicentrarchus labrax was closely related toT, such thatŽ .development stages were earlier completed at higherT Table 1 . Thus, high incubation

Ž . Ž . Ž .Fig. 2. a–c Transverse section of lateral muscle of sea bass. Staining according to Ontell 1974 . a,bOne-day-old Med. larva incubated at 17 and 158C, respectively, bar: 21.11 and 23.045mm, respectively. Athatching larval myotomes showed two muscle layers: a superficial monolayer consisting of small diameterfibres with abundant mitochondria, and a deeper layer of larger fibres that occupied the rest of the myotome.

Ž .These inner fibres still showed early stage of myofibrillogenesis, and nuclei centrally placed. c Six-day Atl.Ž .larva mouth opening reared at 158Crnatural, bar: 11.99mm. At mouth opening increased miofibrilar content

of white muscle fibres. W: white muscle; R: red muscle; n: nucleous of myotube; mf: myofibrils; mi:mitochondria; N: notochord; S: spinal chord.

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Ž . Ž .T 17 8C accelerated embryonic development hatching , and following hatch, higherŽ . Ž .cultivation T 19 8C accelerated prelarval mouth opening , and larval development

Ž .metamorphosis, scaling .

3.2. Muscle growth in the Õitelline phase

Ž .At hatching, higher incubationT 17 8C increased the area of white and red fibres inŽ . Ž .Med. larvae P-0.05 Figs. 2a,b and 3c whereas in Atl. larvae no significantT effect

Ž .was observed at this stage Fig. 3a . At mouth opening, the white muscle fibreŽ . Ž .hypertrophy was greater at 198C than at naturalT in Atl. larvae P-0.05 Fig. 3a ,

Ž .whereas in Med. larvae, these values were similar at allT ’s Fig. 3c . The number ofmuscle fibres remained almost constant and was similar at allT ’s in both populationsŽ .Fig. 3b,d .

(3.3. Growth at larÕal phase: from 20 days until the beginning of inert feeding 48–55)days

During this period, muscle fibre hypertrophy and hyperplasia increased in all groups,Ž . Ž .being greater at 198C than at naturalT P-0.05 Figs. 4a,b and 5 . Furthermore, the

Ž . Ž .Fig. 3. a–d Transverse area of white muscle fibres WMFA values and number of white muscle fibresŽ . Ž . Ž .WMFN values in the differentT regimes at hatching and mouth opening, in Atlantic Atl. sea bass a,b and

Ž . Ž .Mediterranean Med. sea bass c,d . Values represent means"s.e.m., five fish per sample.

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Ž . Ž . Ž .Fig. 4. a–e Transverse section of lateral muscle of sea bass. Staining according to Ontell 1974 . a,bTwenty-day Med. larvae reared at 158Crnatural and 15r19 8C, respectively, bar: 54.25mm. White musclefibres had abundant myofibrils, while the red muscle fibres presented high mitochondrial content around a fewmyofibrils. Distinct germinal zones of muscle fibre were observed at the epi- and hypoaxial limits of the

Ž . Ž . Ž .myotomes. c–e End of metamorphosis scaling : c,d 73-day Med. larva reared at 158Crnatural and 68-dayŽ .Med. larva reared at 178Crnatural, respectively, bar: 36.16mm; e 55-day Med. larva reared at 15r19 8C,

bar: 47.53mm. At scaling small new white muscle fibres were found scattered throughout the myotome. Thisfact is associated with the mosaic appearance of the white muscle. W: white muscle; R: red muscle; nW: newwhite muscle fibres; nR: new red muscle fibres; N: notochord; S: spinal chord; hS: horizontal septum.

( )M.D. Ayala et al.rAquaculture 202 2001 359–370366

Ž .Fig. 5. a–d Effect of rearingT ’s and the incubatingT ’s on the number of white and red muscle fibres inŽ . Ž .Atlantic sea bass a,b and Mediterranean sea bass c,d , at 20 days post-hatch and at the beginning of inert

Ž .feeding 48–55 days . Values represent the means"s.e.m., five fish per sample.

statistical analysis revealed that the effect of the high cultivationT was more significantŽfor the number of fibres than for the area of fibres e.g.Ps0.003 andPs0.044 for

. Ž .number and area of fibres, respectively, in Atl. larvae at 20 days . Body length Fig. 6Ž .and body weight not shown were also statistically higher in larvae reared at 198C

Ž .P-0.05 .

( )3.4. Growth at end of metamorphosis scale appearance

Ž .Metamorphosis was completed at 2"0.5-cm total length in all groups Fig. 6 . Thetotal myotomal area and the number of white muscle fibres in Atl. larvae were higher at

Ž . Ž .19 8C than at naturalT P-0.05 Fig. 7a,c . In Med. sea bass, larvae cultivated at 198C showed a lower size of the myotome than larvae maintained at 178Crnatural and the

Ž .number of fibres was similar at allT Figs. 4c–e and 7d,f . In both populations, theincubationT did not influence muscle cellularity of larvae cultivated at 198C. Thus,muscle parameters were similar at 15r19 8C than at 17r19 8C. On the other hand, in

Ž .tanks reared at naturalT 15 8Crnatural and 178Crnatural , the high incubationT hada positive effect on muscle growth at this stage. Thus, the total myotomal area and the

( )M.D. Ayala et al.rAquaculture 202 2001 359–370 367

Ž . Ž . Ž .Fig. 6. a,b Total length of Atlantic sea bass a and Mediterranean sea bass b from hatching to 90 days.Ž .According to the linear model, growth at the differentT ’s was represented by equations: aLs1.5q0.31t

Ž . Ž 2 . Ž 2 .for 15 8Crnatural tsdays after hatching r s0.97 ; Ls2.03q0.41t for 15r19 8C r s0.99 ; Ls2.28Ž 2 . Ž 2 . Ž .q0.29t for 17 8Crnatural r s0.98 ; Ls1.65q0.38t for 17r19 8C r s0.99 . b Ls2.77q0.27t for

Ž 2 . Ž 2 .15 8Crnatural r s0.99 ; Ls1.17q0.45t for 15r19 8C r s0.97 ; Ls1.88q0.37t for 178CrnaturalŽ 2 . Ž 2 .r s0.98 ; Ls1.58q0.44t for 17r19 8C r s0.94 . Values represent means of 10 fish per sample. Thearrows illustrate the mean time at which the larvae completed metamorphosis, at a body length of 20"5 mm.

( )M.D. Ayala et al.rAquaculture 202 2001 359–370368

Ž . Ž .Fig. 7. a,b Transverse area of total myotomal area TMA values, transverse area of white muscle fibresŽ . Ž .WMFA values and number of white muscle fibres WMFN values determined in the differentT regimes at

Ž . Ž . Ž .the end of metamorphosis scaling in Atlantic sea bass a–c and Mediterranean sea bass d–f . Valuesrepresent means"s.e.m., five fish per sample.

transverse area of white muscle fibres were higher at 178Crnatural than at 15Ž .8Crnatural in both populations Figs. 4c,d and 7a,b,d,e .

Ž .The two-way ANOVA revealed a significative interactionP-0.05 between theincubation and cultivationT ’s on these parameters.

3.5. Growth on postlarÕal phase: 90 days

Ž .White muscle growth not shown , body length and body weight were greater at 19Ž . Ž .8C than at naturalT P-0.05 in both populations Fig. 6 . However, higher mortalities

were observed at 198C. There was no significant effect of incubationT at this moment.

4. Discussion

Ž .As reported for other species Johnston et al., 1996, 1998 , and generally in allectotherms, the rate of development ofD. labrax was closely related withT. Thus,embryonic, prelarval and larval periods were earlier completed at higherT ’s. Musclegrowth varied according to theT regime, but hypertrophy and hyperplasia were notaffected to the same extent. During the vitelline phase, muscle growth was mainly due to

( )M.D. Ayala et al.rAquaculture 202 2001 359–370 369

hypertrophy, and this parameter was more influenced by temperature than hyperplasia.According to these results it seems that hypertrophy is the more efficient mechanism ofmuscle growth during the endogenous feeding period, as was suggested for Usher et al.Ž .1994 in larvae ofS. salar. Similarly, in larvae of C. harengus, the initial muscle

Ž .growth was only due to hypertrophy Johnston et al., 1998 and temperature did notinfluence the number of fibres.

After yolk-sac reabsorption, hypertrophy, and more significantly, hyperplasia weregreater at 198C. Similar results have been reported in larvae of other species, and areconsistent with the general finding that in juvenile fish rapid growth is associated with

Ž .muscle fibre hyperplasia Gibson and Johnston, 1995; Johnston and McLay, 1997 .Ž .When larvae of the same age 20, 48–55 or 90 days were compared, muscle growth

was greater at 198C than at naturalT. However, when comparing larvae at the sameŽ .developmental stage hatching, mouth opening and scaling , such result was not always

Ž .found. Thus, at the end of metamorphosis scaling of Med. larvae, the faster develop-ment of larvae reared at 198C resulted in a lower final size of the myotome than inlarvae maintained at 178Crnatural. The shorter time period between developmental

Ž .stages would thus reduce the total amount of growth Atkinson, 1996 .In both populations of sea bass, muscle growth was higher at 178Crnatural than at

15 8Crnatural at the end of metamorphosis. This fact highlighted a gradual and delayedeffect of the embryonicT on muscle growth, so early developmentT determines futuregrowth characteristics. In other studies, a similar correlation between embryonic temper-

Ž .ature and subsequent larval growth has been found Johnston et al., 1998 .Both populations of sea bass showed, at the end of each developmental stage,

different responses toT in the relative contribution of hypertrophy and hyperplasia. Thismight be associated to the different genetic backgrounds of spawners from Atl. and

ŽMed. origin. Similar situations have been described in other speciesC. harengus,.Johnston et al., 1996;S. salar, Johnston and McLay, 1997 . The variation in muscle

cellularity associated to genetic and environmental factors reveal that hypertrophic andhyperplastic muscle growths show considerable plasticity with respect to extrinsic and

Ž .endogenous factors Nathanailides et al., 1996; Johnston et al., 1998; Johnston, 1999 .These mechanisms are little known and its understanding may be of interest for

Ž .aquaculture in order to improve the production of the fish Johnston, 1999 .

5. Conclusions

Ž .1 High temperature accelerated rate of development and muscle growth, althoughthis was not always accompanied by a higher size of the myotome at the end ofmetamorphosis.

Ž .2 At the end of each stage of development, Atl. and Med. sea bass showed differentmuscle cellularity responses to temperature, which may be associated to geneticsdifferences between them.

Ž .3 The high incubating temperature had a positive effect on muscle growth by the endof metamorphosis, thus indicating that larval growth of sea bass is affected by thethermal conditions experienced by the embryo.

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Acknowledgements

This work was supported by a research grant MAR96-1831 from Spanish CICYT.

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