Amino acid metabolism and protein turnover in larval turbot ( Scophthalmus maximus ) fed natural zooplankton or Artemia

Page 1

L. E. C. ConceicË aÄ o á T. van der Meeren á J. A. J. VerrethM. S. Evjen á D. F. Houlihan á H. J. Fyhn

Amino acid metabolism and protein turnover in larval turbot(Scophthalmus maximus) fed natural zooplankton or Artemia

Received: 19 March 1997 /Accepted: 14 April 1997

Abstract The present paper studied the in¯uence ofdi�erent food regimes on the free amino acid (FAA)pool, the rate of protein turnover, the ¯ux of aminoacids, and their relation to growth of larval turbot(Scophthalmus maximus L.) from ®rst feeding untilmetamorphosis. The amino acid pro®le of protein wasstable during the larval period although some small, butsigni®cant, di�erences were found. Turbot larvae hadproteins which were rich in leucine and aspartate, andpoor in glutamate, suggesting a high leucine require-ment. The pro®le of the FAA pool was highly variableand quite di�erent from the amino acid pro®le in pro-tein. The proportion of essential FAA decreased withdevelopment. High contents of free tyrosine and phe-nylalanine were found on Day 3, while free taurine waspresent at high levels throughout the experimental pe-riod. Larval growth rates were positively correlated withtaurine levels, suggesting a dietary dependency fortaurine and/or sulphur amino acids. Reduced growthrates in Artemia-fed larvae were associated with lowerlevels of free methionine, indicating that this diet is de-®cient in methionine for turbot larvae. Leucine mightalso be limiting turbot growth as the di�erent diet or-ganisms had lower levels of this amino acid in the free

pool than was found in the larval protein. A previouslypresented model was used to describe the ¯ux of aminoacids in growing turbot larvae. The FAA pool wasfound to be small and variable. It was estimated that thedaily dietary amino acid intake might be up to ten timesthe larval FAA pool. In addition, protein synthesis andprotein degradation might daily remove and return,respectively, the equivalent of up to 20 and 10 times thesize of the FAA pool. In an early phase (Day 11) highgrowth rates were associated with a relatively low pro-tein turnover, while at a later stage (Day 17), a muchhigher turnover was observed.

Introduction

Most published work on larval dietary requirements inturbot as in other species focuses on the essential poly-unsaturated fatty acid requirements for survival andgrowth (e.g. Witt et al. 1984; Planas et al. 1993; Reitanet al. 1993; Rainuzzo et al. 1994). However, growth isessentially protein deposition (Houlihan et al. 1993a),and thus an adequate supply of dietary protein is fun-damental for growth optimisation. This protein re-quirement concerns the amount and the quality of thedietary protein, i.e. the balance of the di�erent aminoacids (AA), and in particular the essential amino acids(Tacon and Cowey 1985; D'Mello 1994; Wilson 1994).Fish seem to have the same ten essential amino acids(EAA) as other animals, but tyrosine and cysteine areusually considered semi-EAA, as they can only be syn-thesised from EAA (Wilson 1994). Amino acid imbal-ances will lead to increased AA oxidation and thus todecreased food conversion e�ciencies (growth/food in-take) (Tacon and Cowey 1985). A poor dietary AAbalance also increases the rates of protein synthesis andturnover (Langar et al. 1993). As protein synthesis ishighly energy demanding (Jobling 1985; Houlihan 1991),this will result in lower food conversion e�ciencies. In-fused diets with an imbalanced essential AA composi-tion also give a higher oxygen consumption than balanced

Marine Biology (1997) 129: 255±265 Ó Springer-Verlag 1997

Communicated by O. Kinne, Oldendorf/Luhe

L.E.C. ConceicË aÄ o (&) á J.A.J. VerrethDepartment of Fish Culture and Fisheries,Wageningen Agricultural University, P.O. Box 338,6700 AH Wageningen, The Netherlands

T. van der MeerenInstitute of Marine Research,Austevoll Aquaculture Research Station,N-5392 Storebù, Norway

M.S. Evjen á H.J. FyhnZoological Institute, University of Bergen, Alle gt. 41,N-5007 Bergen, Norway

D.F. HoulihanDepartment of Zoology, University of Aberdeen,Tillydrone Ave., Aberdeen AB9 2TN, Scotland, UK

Page 2

diets (Kaczanowski and Beamish 1996). Since proteinis the most costly component of ®sh diets, it is ofparamount importance for aquaculture to determine theAA pro®le that will minimise the protein requirement ofthe cultured species. This ``optimal'' AA pro®le willdepend on the AA requirement for protein synthesis andthe use of individual AA for energy or other purposes(Rùnnestad and Fyhn 1993; ten Doeschate 1995).

The EAA pro®le of ®sh carcass or muscle is consid-ered to be a good index of EAA requirements in ®sh(Tacon and Cowey 1985; Wilson 1994; Mambrini andKaushik 1995). Changes in the free amino acid (FAA)levels after a meal have also been used as a criterion fordetermining AA requirements, based on the hypothesisthat the free pool concentration of an individual AA willremain low until its requirement is met (Wilson 1994).Furthermore, the use of a model of AA ¯uxes has beenproposed as an e�cient tool for analysing protein andAA metabolism, as it can provide information on re-quirements under di�erent dietary or environmentalconditions (Houlihan et al. 1995a, b).

Fish larvae seem to have a higher protein requirementthan older ®sh (Dabrowski 1986). However, little isknown about AA metabolism and the speci®c AA re-quirements of ®sh larvae after the start of exogenousfeeding. Fiogbe and Kestemont (1995) found thatgold®sh larvae have much higher EAA requirements(g AA g)1 protein) than juvenile and adult ®sh. Rates ofprotein synthesis and turnover have been measured inlarvae of some species in relation to ontogeny, temper-ature or feeding (Fauconneau 1984; Fauconneau et al.1986a, b; Houlihan et al. 1992, 1995c; ConceicË aÄ o et al.1997). Houlihan et al. (1995c) constructed an AA ¯uxmodel for larval herring which suggested that dietaryAA have a limited impact on the pro®le of the FAApool. Furthermore, due to the incomplete developmentof the digestive tract in the early larval stages (Govoni etal. 1986; Segner et al. 1993, 1994), some e�ort has beenmade in recent years to study the importance of FAA indiet utilisation (Fyhn et al. 1993; Rùnnestad and Fyhn1993; Nñss et al. 1995).

The present paper studies the in¯uence of di�erentfood regimes on AA metabolism and protein turnover inrelation to growth in larval turbot (Scophthalmus max-imus L.). Special attention is given to the AA pro®le ofboth protein and the free pool, and to how AA imbal-ances in the diet can a�ect food conversion e�ciency andgrowth. The in¯uence of the experimental food regimeson growth, feeding, cost of growth, and body biochem-ical composition of the same batch of turbot larvae hasbeen analysed (ConceicË aÄ o et al. in preparation).

Materials and methods

Fish and rearing

The turbot (Scophthalmus maximus L.) larvae used in this studywere from the same two groups as described by ConceicË aÄ o et al. (inpreparation). Two groups of 15 000 larvae each were transferred to

two 1500-litre tanks 2 d after hatching. The larvae were reared inthese tanks until Day 26 after hatching, under a natural photope-riod (August, 60°N), at a temperature of 18.0 � 0.3 °C and a sa-linity of 34.3 � 0.2&. Water ¯ow was progressively increasedfrom 0.3 to 1.4 litre min)1, giving a daily exchange rate of therearing volume of 28.8 to 134.4%. To optimise rearing conditionsgreen water was used (Naas et al. 1992). The algae Isochrysisgalbana and Tetraselmis sp. were continuously added to the watersupply in order to maintain a concentration between 50 and100 million cells l)1 in the tanks.

Feeding

Live food was added to the rearing tanks twice a day, in themorning between 0900 and 1100 hrs, and in the afternoon between1600 and 1800 hrs (ConceicË aÄ o et al. in preparation). Daily rationwas calculated from a theoretical bioenergetic model for larvalturbot (van der Meeren 1991). During the ®rst 2 weeks post-hatchall the larvae received both rotifers and natural zooplankton. OnDays 12 to 14, one of the tanks (group ART) received both naturalzooplankton, rotifers and Artemia nauplii, and after that only en-riched Artemia nauplii. The other tank (group ZOO), continued toreceive natural zooplankton until the end of the experiment. Nat-ural zooplankton (mostly nauplii, juvenile and adult stages ofcopepods) was dominated (about 90%, in number) by one species,the copepod Acartia grani. The remaining 10% consisted of amixture of Centropages hamatus, Eurytemora a�nis, harpacticoids,and the cladoceran Evadne normannii.

Sampling

Samples for FAA measurements were taken in the morning onDays 3, 7, 10, 16, 20, 23 and 26 post-hatch, before or shortly afterthe ®rst daily addition of live food to the tanks. Samples for pro-tein-bound amino acids (PAA) were taken in the evening (around1900 hrs) on Days 6, 11, and 23 post-hatch. On Day 11 a secondsample was taken in the morning (0900 hrs). At each samplingpoint (FAA and PAA) pooled samples of 20 (until Day 20) or 10(after Day 20) larvae were taken in four replicates. Samples wererinsed in tap water, dried on a sieve, placed into Nunc cryo tubesand stored at )20 °C until further analysis.

Protein synthesis

Protein synthesis measurements were carried out on Days 11 and17 post-hatch. Larvae were gently transferred to aerated glassbeakers (5 litres) at 18.0 � 0.5 °C, and left to acclimatise for 1 to2 h before measurements started. A solution of 24 mM phenylal-anine in ®ltered sea water containing L-[2,6-3H]phenylalanine(Amersham) at a concentration of 7.4 Mbq ml)1 was prepared, anddiluted 1:50 in sea water. Three groups of ten larvae (Day 11), andsix groups of three larvae (Day 17), were incubated in light for 4 hin about 6 ml of the phenylalanine solution in Nunc multi-welltrays. In order to verify the dynamics of the uptake of labelledphenylalanine from the water and its incorporation into protein, apreliminary experiment was made on yolk-sac larvae close tocomplete yolk absorption (3 d after hatching) with a more exten-sive time course (0, 2, 4, 6 and 8 h).

At the end of each incubation, larvae were put on a planktonnet, rinsed well with distilled water, placed in Eppendorf tubes andfrozen in liquid nitrogen. Each tube (sample) contained one groupof incubated larvae (see above). Samples were kept at )20 °C untilfurther analysis.

The samples were homogenised in 0.5 M perchloric acid andcentrifuged in order to separate the FAA pool from the precipi-tated proteins. Protein content was determined (Lowry et al. 1951)after solubilisation in 0.3 M NaOH. Speci®c activity of protein-bound phenylalanine (Sb, dpm nmol)1 phenylalanine) was calcu-lated as the quotient between liquid scintillation counts (dpm,disintegrations per minute) in solubilised protein and the milli-

256

Page 3

grams of protein added to the scintillation vial divided by 271.4.The latter value refers to the nanomoles of phenylalanine containedin 1 mg of turbot larval protein (see ``Results''). Speci®c activity offree pool phenylalanine (Sa, nmol)1 phenylalanine) was determinedas described by Houlihan et al. (1986). Phenylalanine standardswere included in all determinations and the e�ciency of phenylal-anine recovery was measured (Houlihan et al. 1988). RNA contentswere measured using the dual wavelength method (McMillan andHoulihan 1988) and expressed as RNA/protein ratio (mg RNAmg)1 protein).

Fractional rates of protein synthesis (ks, % d)1) were estimatedusing the ¯ooding dose method equation (Garlick et al. 1980;Houlihan et al. 1988):

ks � �Sb=Sa� � �1=t� � 1440� 100 ;

where Sb is the speci®c activity of protein-bound phenylalanine, Sais the speci®c activity of free pool phenylalanine, t is the time(minutes) from start of incubation, and 1440 is the number ofminutes in one day. Protein degradation (kd, % d)1) can be cal-culated by subtracting protein growth from protein synthesis. In agrowing organism, the rate of protein turnover equals the rate ofprotein degradation (Wiesner and Zak 1991).

Protein growth rates (kg, % d)1) were calculated assuming ex-ponential protein growth (Ricker 1958):

kg � �eg ÿ 1� � 100 with g � �ln�Pro2� ÿ ln�Pro1��=�t2 ÿ t1� ;where Pro2 and Pro1 are the protein contents (mg larva)1) at thetwo nearest sampling points for biochemical composition (Con-ceicË aÄ o et al. in preparation).

RNA e�ciency (kRNA, g protein synthesised g)1 RNA d)1) was

calculated as the ratio between ks and the RNA/protein ratio(Millward et al. 1973).

Amino acid pro®les

The FAA in the samples were extracted for 24 h in 6% (®nalconcentration) trichloroacetic acid. After centrifugation (10 min at15 000 ´g) the supernatants were analysed in an automatic AAanalyser (Chromaspeck J180, Hilger Analytical) with ¯uorimetricdetection (OPA reagent) and high-pressure loading. The amountsof PAA were determined similarly after hydrolysis in 6 M HCl asdescribed by Finn et al. (1995a) on samples which had been ex-tracted for FAA as described above. Plankton samples were alsoanalysed for FAA. These samples were prepared by ®ltration ofknown volumes, with known plankton concentrations, into pre-ashed ®bre glass ®lters (1 lm). All determinations were done withfour replicates. Amino acid pro®les were calculated as the per-centage distribution of the mole contents.

Statistical analysis

Values were given as means � standard deviations. Di�erenceswere considered signi®cant when p < 0:05. Results were analysedthrough the SAS package using one-way ANOVA (Proc GLM),Student's t-test (Proc TTEST), and linear regression (Proc REG)where appropriate.

Results

Amino acid pro®le of protein

The PAA pro®le seemed to be rather stable during thelarval period (Table 1). Small, but signi®cant, di�erenceswere only detected for leucine, lysine, phenylalanine andmethionine. However, the pro®le of the PAA was quitedi�erent from that of the FAA pool. After pooling the®ve sampling points from Table 1, the ``average amino Table1

Scophthalmusmaximus.Comparisonbetweentheaminoacidpro®lesofprotein(PAA)andthefreepool(FAA)ofturbotlarvaeofdi�erentages(dayspost-hatch)andfeeding

regimes.Valuesaregiven

inpercentage(m

olebasis)asmeans�

SDofpooledsamplesof10to

20larvae(n

=4).Signi®cantdi�erencesbetweenPAAandFAAvaluesofeach

amino

acidatthedi�erentdaysare

shownas(t-test):*(p<

0.05),**(p<

0.01),and***(p<

0.001).Signi®cantdi�erencesforthecontentsofeach

aminoacidin

PAAare

shownby

di�erentletters(ANOVA,followed

byaBonferronit-test,p<

0.05)(EAAessentialaminoacids;NEAAnonessentialaminoacids)

Aminoacids

Day6(pm),GroupZOO

Day11(am),GroupZOO

Day11(pm),GroupZOO

Day23(pm),GroupART

Day23(pm),GroupZOO

PAA

FAA

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FAA

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45.5�

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257

Page 4

acid'' in larval turbot protein could be calculated asC4.61H9.30O2.58N1.24S0.02, with a molecular weight of123.8. This average AA pro®le allowed us to calculatethat 1 g of larval nitrogen is equivalent to 6.094 g ofprotein, with 1 mg of protein containing 271.4 nmol ofphenylalanine.

Free amino acids

Within each of the two groups signi®cant, but mostlysmall, di�erences among the di�erent sampling dayswere found for all AA (Table 2). In general, the pro-portions of EAA decreased with development. The moststriking di�erences were the very high proportions oftyrosine and phenylalanine on Day 3 (22.6 and 14.2% ofall AA, respectively), compared to later samples (rang-ing from 3 to 4%). Taurine was the most abundant FAAin all samples, and its contribution to the FAA poolincreased with development (Table 2). Relative c-ami-no-butyric acid (GABA) contents tended to decrease asthe larvae grew.

Larvae fed natural zooplankton (group ZOO) orArtemia (group ART) had small di�erences in the pro®leof the FAA pool on Day 16 post-hatch, but larger dif-ferences were noticed during later development, espe-cially on Days 23 and 26 (Table 2). On Days 23 and 26the proportion of methionine was signi®cantly lower inthe Artemia-fed group, and within this group the me-thionine proportion decreased between Days 23 and 26post-hatch. The relative content of taurine (percentageof all AA) was higher for larvae fed zooplankton thanfor those fed Artemia on Day 20, and the opposite wasfound on Day 26 (Table 2). However, when expressed inabsolute (nmol ind)1) or mass speci®c (nmol lg)1 drywt) amounts, taurine contents were always lower in theArtemia-fed group.

On Day 23 post-hatch some signi®cant variationsbetween the morning (Table 2) and afternoon (Table 1)in the AA pro®le of the FAA pool could be detected inboth groups. The contributions of arginine, lysine andthreonine to the FAA pool in the zooplankton-fedgroup increased during the day, while those of phenyl-alanine and serine decreased. Daily variations in thecontribution of individual AA to the free pool werelarger in the Artemia-fed group, with valine, isoleucine,lysine, threonine, tyrosine and proline increasing duringthe day, and glycine and methionine decreasing. Therewas an increase in the proportion of EAA in the freepool from the morning to the afternoon, and this wasmost noticeable in the Artemia-fed group. However, themethionine proportion decreased in the Artemia-fedgroup. During the course of Day 11 small increases werefound in the proportions of histidine, aspartate andglutamine, while the proportions of isoleucine, tyrosineand glycine decreased (Table 1).

The pro®le of the FAA pool in the food organismswas quite di�erent in the various plankton types (results

not shown). The plankton FAA pro®le was also con-siderably di�erent from the AA pro®le of the larvalprotein (Fig. 1). Highest deviations were observed forthe larger zooplankton on Day 25, followed by the en-riched Artemia and the rotifers. All plankton types an-alysed had a FAA pool which was low in aspartate andleucine, and high in arginine and lysine.

Protein turnover

In a separate incubation with labelled phenylalanine itwas shown that the speci®c activity of phenylalanine inboth the free pool and protein increased linearly withtime (Fig. 2). During the incubations to determine therates of protein synthesis, the free phenylalanine poolwas almost doubled (Table 3). Both the fractional ratesof protein synthesis (ks, % d)1) and protein degradation(kd, % d)1), as well as the fractional rate of proteingrowth (kg, % d)1), were higher for the zooplankton-fedgroup than for the larvae fed Artemia on Day 17 (Ta-ble 3). However, the e�ciencies of retention of theprotein synthesised (ERPS � kg=ks � 100) were higher inthe Artemia-fed group. The rates of protein synthesis,degradation and growth were higher on Day 17 than onDay 11, but the e�ciency of retention of protein syn-thesised decreased (Table 3). On Day 17 the RNA/pro-tein ratio and the RNA e�ciency were higher in thezooplankton-fed group. RNA contents were not mea-sured on Day 11.

Amino acid ¯ux

Flux of AA in turbot larvae was estimated (Fig. 3) basedon the model of Millward and Rivers (1988) and Ho-ulihan et al. (1995a). The values observed for the FAApool (Table 2) and for the rates of protein synthesis,protein degradation and protein growth (Table 3) wereused to calculate the respective ¯ux components. Theprotein pool was calculated using the larval proteincontents (ConceicË aÄ o et al. in preparation) and assumingthat 1 g of protein is equivalent to 8.1 mmol of FAA,according to the average AA calculated in the presentstudy. Amino acid intake values referred to the amountof AA o�ered to the larva per day (see ConceicË aÄ o et al.in preparation). Amino acid losses were calculated asintake minus net growth. It was estimated that turbotlarvae on Day 11 post-hatch deposited (as net growth)only 59% of their AA intake in body proteins, whereason Day 17 larvae fed on zooplankton deposited 93% ofthe AA intake (Fig. 3). The AA losses could then beestimated to amount to 41% of intake at Day 11 and 7%at Day 17. The FAA pool seemed to be 6% of theprotein pool on Day 11, decreasing to 4% on Day 17(Fig. 3). The ¯ux model for the Artemia-fed larvae atDay 17 (not shown) was very similar to the one forlarvae fed zooplankton on the same day.

258

Page 5

Table2

Scophthalmusmaximus.Pro®lesoftheFAApoolofturbotlarvaefednaturalzooplankton(ZOO)orArtem

ia(ART)from®rstfeedinguntilmetamorphosis.Valuesaregiven

inpercentage(m

olebasis)asmeans�

SDofpooledsamplesof10to20larvae(n

=4).EAAandNEAAvaluesarepercentagesofEAA+

NEAA.TaurineandGABAareexpressed

asapercentageofallAA.FAA

gives

thetotalofFAA

content(nmolind

)1).Signi®cantdi�erencesbetweengroupsonthesamedayforeach

aminoacidare

shownas(t-test):

*(p<

0.05),**(p<

0.01),and***(p<

0.001).Signi®cantdi�erencesforthecontentsofeach

aminoacidin

each

grouponthedi�erentdaysare

shownbydi�erentletters

(ANOVA,followed

byaBonferronit-test,p<

0.05)

Amino

acids

Day,group:

3,ZOO

7,ZOO

10,ZOO

16,ZOO

16,ART

20,ZOO

20,ART

23,ZOO

23,ART

26,ZOO

26,ART

leu

6.8�

0.9ab

6.3�

0.2abc4.4�

0.3d

5.4�

0.3cd

5.0�

0.7a

7.0�

0.4a

6.6�

0.8a

5.9�

0.7bc

4.9�

0.4a

6.6�

0.7abc

5.6�

1.1a

lys

3.0�

0.2c

8.9�

0.2b

4.9�

0.4bc

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259

Page 6

Discussion

Amino acid pro®le in protein

The present study suggests small changes in the AApro®le of turbot larvae body proteins during develop-ment (Table 1). Furthermore, Finn et al. (1996) havefound a PAA pro®le for turbot larvae at the end of theyolk-sac stage di�ering considerably from what we ob-served on Day 6 after hatching. The contributions ofalanine and glycine to the PAA pro®le are higher in ourstudy, while isoleucine, valine, methionine and serine aresigni®cantly lower. Compared with the PAA pro®les inturbot eggs (Finn et al. 1996), the di�erences are even

more pronounced. Considerable di�erences in the AApro®le of eggs and whole body have also been reportedfor other ®sh species (Ketola 1982; Wilson and Poe1985; Ng and Hung 1994). However, it is often acceptedthat AA pro®les of the whole body change little betweenand within species (Wilson and Poe 1985; Ramseyer andGarling 1994; Wilson 1994). Nevertheless, signi®cantdi�erences in AA pro®les during development have alsobeen observed in dolphin ®sh (Ostrowski and Divakaran1989), white sturgeon (Ng and Hung 1994) and Africancat®sh (ConceicË aÄ o et al. unpublished data). This varia-tion in AA pro®les is probably a result of di�erences inthe relative importance of individual proteins. Com-pared to other species, turbot larvae have proteins withconsiderably higher amounts of leucine and aspartate,and lower amounts of glutamate. Therefore, the re-quirement for leucine may be higher in turbot larvaethan in other ®sh species.

Fig. 1 Di�erence between amino acid pro®les of Scophthalmusmaximus larval protein (Table 1) and of the free amino acid (FAA)pool of the plankton organisms used as food: rotifers Day 10 (h);Artemia Day 25 (s); natural zooplankton Day 9 (r); naturalzooplankton Day 18 (.); small natural zooplankton Day 25 (d); bignatural zooplankton Day 25 (n) (EAA essential amino acids; NEAAnonessential amino acids)

Fig. 2 Scophthalmus maximus. Time course of the speci®c activities ofphenylalanine in the protein (Sb) and in the free pool (Sa) in thepreliminary incubation. Values are means � SD of pooled samples of30 to 40 larvae. Regression lines are also given �Sb � 0:94� 2:95� t,r2 � 0:93, p < 0:0001; Sa � 55:0� 92:1� t, r2 � 0:87, p < 0:0001�

Table 3 Scophthalmus maximus. Fractional rates of proteinsynthesis �ks�, growth (kg), and degradation (kd), together with thespeci®c activities of phenylalanine in the free pool (Sa) and inprotein (Sb) in turbot larvae. E�ciencies of retention of the syn-thesised protein (ERPS ), RNA/protein ratios, and RNA e�-ciencies �kRNA� are also given. Free phenylalanine pools (Free Phe)

are given for larvae before (BI ) and after (AI ) the incubation.Values are means � SD of pooled samples of 10 larvae (n = 3) onDay 11, and 3 larvae (n = 6) on Day 17. Values with di�erentletters in the same row are signi®cantly di�erent (ANOVA, fol-lowed by a Bonferroni t-test, p < 0.05)

Day, group:

11, ZOO 17, ZOO 17, ART

Sa (dpm nmol)1) 1710.4 � 128.9 1006.8 � 9.3 1519.2 � 166.5Sb (dpm nmol)1) 47.1 � 1.4 39.7 � 5.3 62.0 � 11.0ks (% d)1) 33.0 � 2.8 b 69.7 � 9.9 a 44.2 � 7.4 bkg (% d)1) 31.0 41.6 32.6kd (% d)1) 2.0 28.1 11.6ERPS (%) 93.9 � 8.0 a 59.7 � 7.8 c 73.7 � 11.6 bRNA/protein (lg mg)1) 48.8 � 2.1 a 42.9 � 0.8 bkRNA (g g)1 d)1) 14.3 � 1.7 a 10.3 � 1.8 bFree Phe (BI) (nmol) 1.1 � 0.2 3.4 � 0.4 1.8 � 0.3Free Phe (AI) (nmol) 2.0 � 0.1 6.6 � 0.6 2.4 � 0.5

260

Page 7

E�ects of free amino acids on growthand development

The losses of EAA through catabolism are probablyreduced during development by a decrease in the frac-tion of EAA in the free pool. The a�nity of the enzymesinvolved in protein synthesis for AA is higher than thatof the enzymes involved in AA catabolism (Cowey andWalton 1989) and thus lower concentrations of EAAshould allow for a lower oxidation of EAA. Interest-ingly, based on the AA ¯ux diagrams (Fig. 3) the lossesof AA seem to have been much higher on Day 11 thanon Day 17.

The contribution of tyrosine and phenylalanine to theFAA pool on Day 3 post-hatch (22.6 and 14.2% of allAA, respectively) is high when compared to later sam-ples in this study (ranging from 3 to 4%). Finn et al.(1996) have also found the relative contents of tyrosineand phenylalanine to increase towards complete yolk-sacabsorption in turbot larvae, reaching up to 16 and 7% ofthe total FAA pool, respectively. There seems to be atemporary rise in the proportions of these two aromaticAA in the FAA pool around ®rst feeding. Such an in-crease in the relative contents of tyrosine and phenylal-anine close to complete yolk absorption has also beenobserved in gilthead sea bream (Rùnnestad et al. 1994)but not in Atlantic cod (Finn et al. 1995a). Phenylala-nine is the precursor for the synthesis of tyrosine, andthis AA makes up the carbon-skeleton of the thyroidhormones, melanin, dopamine and catecholamines(Bender 1985; Cowey and Walton 1989). It is temptingto suggest that the rise in tyrosine (and phenylalanine) isrelated to the start of the activity of the thyroid gland.Thyroid follicles in the larvae of pelagic marine ®sh ®rstappear just before complete yolk absorption (Tanakaet al. 1995). Marked increases in thyroid hormone levelsaround ®rst feeding have been observed in two salmonspecies (Kobuke et al. 1987; Tagawa and Hirano 1987)and in striped bass (Brown et al. 1988).

High levels of taurine in the FAA pool, comparableto those observed in the present study, have been ob-served in yolk-sac larvae of turbot (Finn et al. 1996),Atlantic cod (Finn et al. 1995a) and Atlantic halibut

(Finn et al. 1995b). Such high taurine contents are alsocommon in the FAA pool of tissues, but not plasma, ofjuvenile ®sh (e.g. Walton and Wilson 1986; Cowey andWalton 1989; Lyndon et al. 1993; Carter et al. 1995) andmammals (Hayes and Sturman 1981). The function ofthese high levels of taurine is not well understood, al-though taurine is known to be the sole AA that conju-gates with cholic acid to produce the bile salts in teleost®shes (van Waarde 1988). In addition, taurine seems tobe an important osmolyte involved in cell volume reg-ulation in ®sh (Fugelli and Zachariassen 1976; Vislie1982), and takes part in a series of neuronal and mem-brane-related functions (Hayes and Sturman 1981;Huxtable 1992). In animals, taurine cannot be brokendown for energy (Huxtable 1992). Taurine is synthesisedfrom methionine via cysteine. High levels of cysteine ormethionine in the diet increase taurine production in therat (Tanaka et al. 1993; Yamada et al. 1995). The sameseems to be true for rainbow trout (Yokoyama andNakazoe 1989). Based on data for skate, it has beensuggested that ®sh are unable to synthesise taurine(Cowey and Walton 1989). If this is the case for turbotlarvae, there may be a speci®c dietary requirement fortaurine, as has also been found for the cats (Hayes andSturman 1981).

The present study suggests a correlation betweentaurine levels and growth rates of turbot larvae. The lowgrowth rate observed in group ART at the end of theexperiment (ConceicË aÄ o et al. in preparation) was ac-companied by lower taurine contents than in groupZOO. Furthermore, turbot larvae growing at lower ratesdue to food limitation have also had lower taurinecontents (ConceicË aÄ o et al. unpublished data). This in-dicates that an insu�cient dietary supply of taurine,and/or its precursors methionine and cysteine, may re-duce growth in turbot larvae.

Between Day 23 and Day 26 post-hatching, Artemia-fed turbot larvae (group ART) had lower growth rates(16.6% body dry wt d)1) and higher lipid deposition(19.5% dry matter) than group ZOO (25.6% body drywt d)1 and 14.1% lipids in dry matter) (ConceicË aÄ o et al.in preparation). This may be related to a suboptimaldietary quality of the enriched Artemia, which may be

Fig. 3 Scophthalmus maximus. Amino acid ¯ux for turbot larvae aDay 11 and b Day 17 post-hatch and fed natural zooplankton, basedon the model of Millward and Rivers (1988). Values observed for theFAA pool (Table 2) and for the rates of protein synthesis, proteindegradation and protein growth (Table 3) were used. According to the

average amino acid pro®le calculated in this study 1 g of proteinequivalent to 8.1 mmol of free amino acids. Intake values refer toamount of amino acids o�ered to the larva per day (ConceicË aÄ o et al.in preparation). Amino acid losses were calculated as intake minus netgrowth

261

Page 8

de®cient in methionine for turbot larvae. The contribu-tion of methionine to the FAA pool on Days 23 and 26was lower for the Artemia-fed larvae, and also decreasedbetween Days 23 and 26 within this group (Table 2).Furthermore, on Day 23, while generally the Artemia-fed group had a higher increase in EAA during the day,methionine was the sole EAA which decreased signi®-cantly. The methionine content in Artemia protein(Seidel et al. 1980) is considerably lower than that ob-served in turbot larvae protein (Table 1).

Another AA which may limit growth in turbot larvaeis leucine. The FAA pool in the di�erent plankton or-ganisms used in this study, and in Artemia in particular,is poor in leucine when compared to the content ofleucine in larval protein (Fig. 1). Comparing with liter-ature values (Seidel et al. 1980), the leucine level in Ar-temia protein is also considerably lower than that foundin larval turbot protein (Table 1). The AA pro®le of thedietary plankton food seems rather imbalanced if theFAA pro®le of the plankton organisms is representativefor the assimilated AA in the gut (Fig. 1). However it isdoubtful whether this is the case as the AA in protein arequantitatively much more important than FAA (Con-ceicË aÄ o et al. in preparation). In rotifers, unenriched Ar-temia and in various freshwater zooplankton organisms,large di�erences have been found between the AA pro-®les in protein and in the FAA pool (Dabrowski andRusiecki 1983).

The pro®le of the FAA pool in turbot larvae is quitevariable. The daily and the day-to-day variations(Tables 1, 2) may be attributable to dietary in¯uences. Agood correlation between the individual AA levels ofdietary EAA and plasma EAA has been found in juve-niles and adults of several ®sh species (Plakas et al. 1980;Wilson et al. 1985; Walton and Wilson 1986; Lyndon etal. 1993; Kaushik et al. 1994; Schuhmacher et al. 1995).No correlation has been found in these studies betweendietary and plasma NEAA. On the contrary, liver andmuscle levels of free EAA show a poor correlation withdietary EAA (Walton and Wilson 1986; Lyndon et al.1993; Carter et al. 1995). However, the variations in theAA pro®les from samples taken in the morning(Table 2) should be relatively diet independent, sincefood intake during this part of the day seems to be low inturbot larvae (Danielssen et al. 1990). Therefore, thevariability in the pro®le of the FAA pool also seems tobe a�ected by developmental events and physiologicalconditions.

Protein synthesis and turnover

The measurements of protein synthesis met the criteriafor successful measurements with the ¯ooding dosemethod (Garlick et al. 1980; Houlihan et al. 1988). Therewas a linear labelling of protein with time in larvalturbot (Fig. 2). Taking into account the fast increase inthe speci®c activity of the free pool of phenylalanine andthe high growth rate of the larvae, labelled phenylala-

nine was assumed to be homogeneously distributedamong the di�erent metabolic pools. The comparison ofthe protein synthesis rates with the rates of proteingrowth, and subsequent calculation of the protein de-gradation rate, is somewhat uncertain due to the dif-ference in time scale of the two measurements (Houlihanet al. 1995a). In the present study, synthesis rates weremeasured over a period of 2 to 4 h, while growth rateswere measured over a period of 4 to 6 d.

Results on Day 17 post-hatch (Table 3) support thegeneral hypothesis that higher protein growth rates areassociated not only with increased rates of protein syn-thesis but also with higher protein turnover (Waterlowet al. 1978; Houlihan et al. 1986, 1988). However, un-changed rates of protein turnover at di�erent proteinsynthesis rates have been observed in larval nase (Ho-ulihan et al. 1992) and have also been suggested for yolk-sac larvae of the African cat®sh (ConceicË aÄ o et al. 1997).

Interestingly, the e�ciencies of retention of the pro-tein synthesised are reduced with increasing proteinsynthesis rates (Table 3). The e�ciency on Day 11 issurprisingly high compared to published values for ®shlarvae (Houlihan et al. 1992, 1995c; ConceicË aÄ o et al.1997). It is the ®rst experimental support for the sug-gestion that ®sh larvae may reduce their levels of proteinturnover in order to save energy for growth (Kiùrboeet al. 1987; Kiùrboe 1989; Wieser and Medgyesy 1990;Wieser 1994).

The RNA/protein ratios are comparable to valuesnormally reported for ®sh larvae (about 45 lg RNAmg)1 protein) which are much higher than values foradult ®sh (Houlihan et al. 1995b). However, the RNAe�ciencies are high when compared to published values,typically ranging from 3 to 6 g protein synthesisedg)1 RNA d)1 (Houlihan et al. 1995b). ComparableRNA e�ciencies have only been found in 10-mg tilapia(Houlihan et al. 1993b), in yolk-sac larvae of the Africancat®sh (ConceicË aÄ o et al. 1997) and in mammals (Reedsand Davies 1992). High protein synthesis rates in larvalturbot are obtained through very high RNA e�ciencies,associated with high RNA concentrations.

Amino acid ¯ux and growth

The AA ¯ux diagram for Day 17 (Fig. 3b) suggests avery high protein conversion e�ciency (growth/intake)compared to the 63% found in larval herring (Houlihanet al. 1995b), and values between 20 and 48% found injuvenile and in adult ®sh (Bowen 1987; Houlihan et al.1995a). Protein conversion e�ciencies of larval ®sh arenot available in literature, probably due to the di�cul-ties in measuring food (and protein) intake. The esti-mates of protein conversion e�ciencies in the presentstudy should be treated with caution, as AA intake isestimated based only on the food o�ered to the larvae.The estimates of AA losses are also dependent on theuncertainties of AA intake. Amino acid losses includeboth AA oxidation and AA which are eaten but not

262

Page 9

assimilated in the gut. Conway et al. (1993) found thatthe AA assimilation e�ciency of di�erent zooplanktonorganisms is close to 100% in turbot larvae with an ageof 21 to 27 d post-hatch. However, in the present studythe larvae were younger, and therefore the digestivesystem may have been less developed. Striped bass lar-vae have been found to have protein assimilation e�-ciencies increasing from 30% in young stages to 60%around metamorphosis (Rust 1995).

The AA ¯ux diagram for Day 11 (Fig. 3a) is com-parable to the one published for larval herring (Houli-han et al. 1995b), except in what concerns the size of thefree pool. On Day 11 the size of the FAA pool is 6% ofthe protein pool (4% on Day 17) compared to 29% inherring larvae. In this respect our results are closer to thevalues around 2.3% found in juvenile rainbow trout(Carter et al. 1995; Houlihan et al. 1995a, b). This meansthat on a daily basis the dietary AA supply may be up toten times the larval FAA pool. In addition, proteinsynthesis will daily remove the equivalent of 5 (Day 11)or 20 (Day 17) times the size of the FAA pool. Similarly,protein degradation will return to the FAA pool lessthan 1 (Day 11) or 10 (Day 17) times its size. So, theFAA pool in growing turbot larvae is extremely dy-namic, being very sensitive to the arrival of dietary AA,as well as to the AA pro®les of proteins that are beingsynthesised and/or degraded at a given moment. Thehigh variation in the pro®les of the FAA pool supportsthis idea.

The present study suggests that there are some im-portant ontogenic changes in AA metabolism in larvalturbot. In an initial phase (Day 11 post-hatch), highgrowth rates seem to involve a reduction in the turnoverof proteins, while AA losses through oxidation appear tobe high. This high AA oxidation may be related to thehigh larval FAA concentration when compared witholder larvae (see above). Lower protein turnover maysave energy for growth (or other processes) as proteinturnover is responsible for a large fraction of the energybudget (Hawkins et al. 1989; ConceicË aÄ o et al. 1997).Furthermore, a reduced protein turnover rate is alsolikely to diminish losses by oxidation of the AA involvedin turnover (Carter et al. 1995). However, it has beenproposed that reducing protein turnover may have costsfor the larvae in terms of viability and survival (Kiùrboeet al. 1987). High protein turnover will allow for moremetabolic plasticity, enabling a fast response of the or-ganism to environmental/disease stress, through thesynthesis of speci®c enzymes and other proteins.

At a later stage (Day 17 post-hatch), a much higherprotein turnover is observed, but the oxidative losses ofAA seem to be much smaller. During development,turbot larvae may acquire the capacity for (down) reg-ulation of the catabolism of AA. Dabrowski (1986,1989) proposed a decreasing role of AA catabolism forenergy production during ®sh development. Further-more, the activities of enzymes involved in AA catabo-lism are reduced with increasing size in larvae of theAfrican cat®sh (Segner and Verreth 1995). This suggests

that an eventual increase in AA oxidation associatedwith higher protein turnover rates can be minimised. Insuch conditions, investment of energy in protein turn-over may be a bene®cial strategy that increases viability.Older larvae may also attempt to optimise the AA re-sources by reducing both the size and the proportion ofEAA in the FAA pool.

The AA pro®le of the dietary protein may also a�ectthe AA ¯ux and AA utilisation e�ciency. As larvae canonly store AA in the form of proteins, imbalances be-tween dietary and larval AA pro®les will tend to bringan unavoidable AA loss. Poor dietary AA balance hasalso been shown to increase the rates of protein synthesisand turnover (Langar et al. 1993). However, dietary AAimbalances may be compensated by the endogenousrelease of free AA through protein turnover. In addition,the absorption of individual AA in the gut may proceedat di�erent rates (Dabrowski 1986). Therefore, variationin the rates of absorption of individual AA may atten-uate or aggravate AA losses. It would be interesting toinvestigate the e�ect of diets with di�erent AA pro®leson larval AA ¯ux.

Acknowledgements We thank T. F. Galloway for revising themanuscript. L. ConceicË aÄ o participated in this work with a grantfrom the Programa PRAXIS XXI (Portugal).

References

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