Aquaculture SEP 2004; 238(1-4) : Copyright 2004 Elsevier B.V. All rights reserved

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1 Aquculture SEP 2004; 238(1-4) : Copyright 2004 Elsevier B.V. All rights reserved Archimer Archive Institutionnelle de l Ifremer Expression nd ctivities of pncretic enzymes in developing se ss lrve (Dicentrrchus lrx) in reltion to intct nd hydrolyzed dietry protein; involvement of cholecystokinin C. Chu,*, I. Rønnestd, V. Grngier nd J. L. Zmonino Infnte : INRA-IFREMER Twin-Unit of Fish Nutrition, IFREMER, B.P. 70, Plouzné, Frnce : Deprtment of Zoology, University of Bergen, Allégt 41, N5007, Bergen, Norwy *: Corresponding uthor : E-mil: cchu@ifremer.fr, Fx: Astrct: In order to ssess the influence of dietry protein on digestive enzyme expression nd cholecystokinin (CCK) content in se ss lrve, four groups of lrve were fed experimentl diets from mouth opening until dy 42: three isonitrogenous diets with incresing protein hydrolyste levels (0%, 14% nd 46% of crude mtter) nd one diet incorporting strch. The groups fed high strch or high protein hydrolyste level exhiited the lowest growth. The finl weight in these groups ws 9.5 nd 5.6 mg, respectively, wheres it reched pproximtely 20.0 mg in the groups fed 0% or 14% protein hydrolyste level. The highest levels of trypsin secretion were oserved in lrve fed the lowest protein hydrolyste level. Prdoxiclly, the groups fed diet contining strch lso exhiited high trypsin secretion level. There ws cler llometric reltionship etween lrvl CCK content nd ody mss, ut there were lso differences etween dietry groups. On dy 42, the CCK level in the group fed diet incorporting strch (40 fmol/mg dry weight of lrve) ws more thn twice s high s tht found in the other groups. The lowest CCK level (13 fmol/mg) ws found in the group fed the highest protein hydrolyste level. Our dt suggested tht dietry protein level nd chin length comined with protein intrluminl proteolytic ctivity regulte the CCK level in fish lrve s in other vertertes. The CCK concentrtion ssyed in lrve fed diets with low protein content or different protein hydrolyste levels is comptile with the existence of n indirect mechnism controlling CCK relese nd mediting pncretic enzyme secretion. Keywords: Cholecystokinin; Pncretic enzyme secretion; Protein hydrolyste; Se ss lrve; Strch 1

2 2 1. Introduction Mrine fish lrve re not completely developed t htching nd they undergo mjor morphologicl nd functionl chnges during the first weeks of life. A proper development of the digestive trct is essentil for the trnsition from n endogenous to exogenous feeding (Boulhic nd Gudn, 1992; Wlford nd Lm, 1993). The pncres cquires progressively its enzymtic cpcity (Krogdhl nd Sundy, 1999) nd its secretory function (Beccri et l., 1991). Recent studies demonstrted tht the developmentl process of pncres could e ffected y the composition of the exogenous diet (Zmonino Infnte nd Chu, 1994; Perés et l., 1996). In dult fish nd in mmmls, cholecystokinin (CCK) plys mjor role in the pncretic enzyme secretion together with neurl stimultion (Singer 1993). It is produced in specific cells scttered in the mucos of the proximl intestine nd is secreted into the plsm in response to the presence of nutrients in the lumen (Liddle, 1997). The existence of n endogenous trypsin-sensitive luminl CCK relesing fctor ws demonstrted in 1989 in rts (Liddle, 1995). This fctor is modulted y the nture nd chin length of dietry protein (Owyng, 1994), which intercts with nd regultes the CCK-secreting cells of the intestine (Liddle, 1995). In rts, CCK secretion is more stimulted y intct proteins thn hydrolyzed proteins or mino cids (Green nd Miysk, 1983), while it is less stimulted y crohydrtes thn ft or proteins (Liddle et l., 1986). It is not known when these mechnisms controlling pncretic secretion ecome effective in developing nimls. Mrine fish lrve constitute n interesting niml model since they cquire progressively their dult mode of digestion. There re very few studies on CCK in fish lrve, nd reserch on this topic is still in its infncy. CCK-producing cells hve een

3 3 detected from the first dy post-htch in Jpnese flounder, Prlichthys olivceus (Kurokw et l., 2000), tun, Thunnus thynnus, (Kmisk et l., 2002) nd yu, Plecoglossus ltivelis (Kmisk et l., 2003), while CCK hs not een detected in the gut t the onset of exogenous feeding in Atlntic hliut, Hippoglossus hippoglossus, (Kmisk et l., 2001, Rojs-Grcí nd Rønnestd, 2002). Nevertheless, in older nimls, short term experiments performed with tue-fed lrve hve strongly suggested the involvement of this hormone in protein digestion in post-lrvl Atlntic hliut (Rojs-Grcí nd Rønnestd, 2002) nd lso in herring lrve from onset of exogenous feeding (Koven et l., 2002). Our experiment imed to study the expression of two mjor pncretic enzymes nd to ssess the involvement of CCK in the regultion of their secretion, during the development of se ss lrve fed different dietry protein concentrtion nd chin length. 2. Mterils nd methods 2.1. Animls nd diets Eggs of Europen se ss (Dicentrrchus lrx) were otined from Aqunord. The lrve were rered t the Ifremer-Sttion de Brest during 42 dys, period which corresponds to the end of lrvl development. Newly htched lrve were trnsferred from incutors to 16 conicl fier-glss tnks (35 L) with lck wlls (initil stocking density: 60 lrve L -1, i.e lrve per tnk). They were supplied with running se wter (20 C; slinity: 35 g L -1 ), which hd een filtered through snd filter, then pssed successively through tungsten heter nd degssing column pcked with plstic rings. Wter exchnge rte ws up to 30% per hour (flow rte: 0.18 L min -1 ) to mintin oxygen level ove 6 mg L -1. The light intensity ws mximum of 9 W m -2 t the surfce. All niml procedures nd hndling were conducted in complince with the Guide for the Cre nd Use of Lortory Animl (NRC 1985).

4 4 Lrve were divided into four groups (four tnks per group) efore mouth opening (d 6 post-htching). They were fed from d 6 to d 42 post-htching on one of four diets (Tle 1): three isonitrogenous diets with incresing protein hydrolyste levels (NAP, LHY nd HHY) nd one diet incorporting strch (SAP). The size of the dietry microprticles ws µm during the first 5 d, then µm. Fish were continuously fed to lrge excess for 18 hours per dy y mens of elt feeder. Lrvl digestive trcts were oserved under inoculr microscope in order to monitor food ingestion, since microprticles re visile through the trnsprent lrve Smplings nd dissection Wter volume in tnks ws reduced nd ten lrve (n=4 tnks for ech dietry group) were tken from ech tnk in one single stroke using net, for determintion of wet mss t the eginning nd the end of the experiment. These lrve were representtive of the tnk popultion. At the end of the experiment, the remining lrve were counted to determine survivl rtes. On d 25 nd d 42, lrve were collected from ech tnk for enzymtic studies (50 lrve) or CCK determintions (15 lrve) efore the morning food distriution; they were immeditely stored t -80 C pending dissection nd ssys or lyophiliztion nd shipment to Norwy. Fifty more lrve were collected for mrna studies from only three tnks per dietry tretment were dissected nd RNA were immeditely extrcted. Lrve were dissected into four prts under microscope on glss slide mintined t 0 C; s descried y Chu nd Zmonino Infnte (1994): hed, pncretic segment, intestinl segment nd til, in order to limit the ssy of enzymes to specific segments. The dissection

5 5 enles distinguishing etween pncretic (synthesized enzymes) nd intestinl (secreted enzymes) ctivities (Zmonino Infnte et l.1997) Anlyticl methods The pncretic nd intestinl segments were homogenized into five volumes (v/w) of icecold distilled wter. Amylse (EC ) ctivity nd trypsin (EC ) were ssyed ccording to Métis nd Bieth (1968) nd Holm et l. (1988) respectively. Enzyme ctivities were expressed s specific ctivities, i.e. mu mg protein -1 Secretion of pncretic enzymes ws clculted s follows: enzyme ssyed in the intestinl segment (mu/segment) divided y enzyme ssyed in pncretic plus intestinl segment (Zmonino Infnte nd Chu, 1999). Protein ws determined ccording to Brdford (1976). Reverse trnscriptse-polymerse chin rection (RT-PCR) nlysis. Totl RNA ws extrcted using the TRIzol regent kit procedure (Life Technologies, Grnd Islnd, NY). For synthesis of cdna, 5 µg of totl RNA ws treted with FPLCpure Moloney-Murine Leukemi virus reverse trnscriptse using the Redy-To-Go-T-Primed First Strnd Kit (Phrmci Biotech, Uppsl, Sweden). PCR ws crried out y n initil denturtion t 94 C for 1 min, followed y 30 cycles t tempertures of 94 C for 30 sec, specific nneling tempertures for 1.5 min, nd 72 C for 1 min; the finl extension ws conducted t 72 C for 7 min. The PCR mixture contined 0.8 µl of the cdna, 0.2 Units Tq Polymerse (Appligene, Githersurg, MD), 100µmol/L dntp, 50pmol of ech primer nd 1X Rection Buffer (Appligene), in finl volume of 50µL. Sequences nd nneling tempertures for the sense nd ntisense oligonucleotides were s follows: 5 - GCCATCAATGACCCCTT-3 nd 5 -GGTGCAGGATGCATTGC-3 (50 C) for glycerldehyde-3-phosphte dehydrogense (GAPDH), 5 -AAGCACATGTGGCCY-3 nd

6 6 5 -CCAGCGCCACTCRAA-3 (54 C) for mylse nd 5 -CAGGTGTCTCTGAAC-3 nd 5 -CCCARGACACAACACCCTG-3 (60 C) for trypsin, respectively. These primers were selected fter lignment of sequences from different species of selected mrna, otined using the Sequence Retrievl System WWW server t EMBL-EBI (Cmridge, UK). Alignments of cdna sequences of species from different phyl were necessry s fish RNA sequences were scrce. The resulting PCR products were cloned using the TOPO-TA Cloning Kit (Invitrogen, Leek, The Netherlnds) nd sequenced y Cyergene (St Mlo, Frnce); the glycerldehyde-3-phosphte dehydrogense (GAPDH), mylse nd trypsin sequences otined hve een registered y EMBL under ccession numers AJ006883, AJ nd AJ respectively. GAPDH, mylse nd trypsin were seprtely mplified using the sme cdna smple; 1 to 10 µl of ech PCR product were pplied on 1.2% grose-1mg/l ethidium romide gel. cdna nds were quntified using Fluor-S TM Multi-imger nd its MultiAnlyst Softwre (BIORAD, Hercules, CA), using n pproprite clirtion (Freemn et l., 199). We generted for ech gene stndrd curve y plotting the UV sornce of the nds, resulting from 30 cycle PCR, ginst known strt concentrtion of the studied cdna (using t lest 7 different dilutions of the studied cdna). We determined the limits of the exponentil phse nd the eginning of the sturtion phse of the mplifiction rection; this enled us to ensure tht there ws liner reltionship etween input RNA nd finl RT- PCR product, or to mintin it y n pproprite dilution of the input cdna. The numer of cycles hs een optimized in order to produce esily visulized products nd to remin out of the plteu phse of the rection. We lso sequenced the mplified cdna products otined in gels in order to control they were derived from mylse nd trypsin.

7 7 The vlues of cdna otined were normlized reltive to the GAPDH cdna, y clculting the mylse/gapdh nd trypsin/gapdh rtios. Indeed, Sölch nd Arnold (1996) hve shown tht this normliztion reltive to GAPDH provides widely pplicle vlue for comprtive studies of gene expression t the mrna level. Rdio-immunossy (RIA). Lyophilized individul lrve were trnsferred to tred Eppendorf tues nd weighed; CCK ws susequently extrcted in methnol s descried y Rojs-Grcí et l., (2001). The methnol superntnts were evported nd the remining dried extrcts nlysed for CCK y competitive rdio-immunossy (RIA) kit (EURIA-CCK, Euro-Dignostic, Mlmö Sweden) using non-piscine ntiserum nti-cck-8 with high specificity for sulphted forms of CCK-8, CCK-22, CCK-33 nd CCK-58 (Rehfeld 1998). CCK levels in the smples were clculted in pmol L -1 y interpoltion from the stndrd curve with corrections for nonspecific inding nd recovery determined for every run. CCK is presented s fmol ind -1 nd fmol mg -1 Dry Mtter (+ SD) 2.5. Sttisticl nlyses. Results re given s men + SD (n = 4; n = 3 for mrna studies; n = 4 to 6 for CCK). Survivl rtes, mlformtion rtes nd rtios of mrna were rcsin(x 1/2 ) trnsformed. The vrince homogeneity of the dt ws checked using Brtlett s test (Dgnelie, 1975). Weight, CCK, survivl rte nd rtios of enzymtic segmentl ctivity dt were compred y onewy ANOVA followed y the Newmn Keuls multiple rnge test (Dgnelie, 1975) when significnt differences were found t the 0.05 level. 3. Results

8 Rering performnces The microdiets were efficiently ingested y the lrve of the four dietry tretments from strting exogenous feeding. Lrvl growth ws more thn two times s high in groups fed diets tht incorported ntive protein (NAP) nd low hydrolyste levels (LHY) s in the two other groups (Tle 2). Survivl on dy 42 ws significntly lower in the group fed high hydrolyste level diet (HHY) Enzyme expression Lower trypsin ctivity levels (Tle 3) were oserved in the homogentes of pncretic segment on dy 25 s well s on dy 42 in the group fed diet incorporting strch nd low protein level (SAP). At dy 42, only the two diets incorporting high percentge of ntive protein (NAP nd LHY) induced high trypsin ctivity. In the sme wy, diets NAP nd LHY lso induced high level of trypsin-coding RNA on dy 42. The lowest level of trypsin ctivity ssyed in intestinl segments ws oserved in the group fed the SAP diet on dys 25 nd 42 (Fig. 1). The highest mylse ctivities were recorded on dy 25 nd dy 42 in the group which ws fed the diet incorporting strch (SAP). Differences in mylse/gapdh rtios only ppered on dy 42: groups fed SAP nd HHY exhiited the highest rtios, while the NAP group showed the lowest (Tle 3) Vrition in CCK relted with vritions in pncretic secretion There ws n llometric increse in lrvl CCK content with development. Expressed on weight-specific sis the CCK content displyed significnt (second-order polynomil ANCOVA using dry weight s covrite, f=64.05; p<0.0001) decline with development (Fig. 2). On dy 25, the highest level of CCK ws ssyed in the NAP nd LHY groups (Fig 3A);

9 9 when expressed s fmol. mg -1 dry weight, high level of CCK ws found in the SAP group (Fig. 3B). On dy 42, the CCK level/mg dry weight in the group fed the SAP diet ws more thn twice s high s tht found in fish of the other dietry groups collected on the sme dte (Fig. 3B). The lowest CCK level ws found in the group fed HHY diet. The sme trend ppered when CCK ws expressed in terms of fmol per lrve. The highest levels of trypsin secretion were oserved in lrve fed the highest levels of ntive protein (NAP nd LHY) nd the lowest level ws oserved in the group fed high level of hydrolyste (HHY). Prdoxiclly, the group which ws fed SAP exhiited level of trypsin secretion very much higher thn tht of the HHY group (Fig. 3C). On dy 25, mylse secretion ws the highest in the SAP group, while on dy 42, the HHY group exhiited rte of mylse secretion significntly lower (P<0.05) thn tht of the three other groups (Fig. 3D). 4. Discussion The purpose of this experiment ws to investigte the mechnisms tht regulte exocrine pncretic secretion, in prticulr, the involvement of CCK, during se ss development. Nutrient delivery into the intestinl lumen is the most importnt stimuli of exocrine pncretic secretion (Liddle et l., 1986). Digestive products of dietry protein, i.e. protein hydrolystes nd mino cids, modulte the ction of pncretic proteses, nd prticulrly trypsin, which in turn control CCK relese in mmmls (Owyng, 1994). We decided to formulte 3 isoproteic diets contining different mount protein hydrolystes nd evlute the consequent effect on CCK relese nd pncretic secretion. An dditionl diet contining low level of protein ws lso considered in order to ssess the influence of the dietry protein content. In this diet, SAP, the lowering in protein level ws compensted in energy y moderte supply of strch. Indeed, previous study demonstrted tht 17% strch incorportion in diet llowed good growth nd survivl in se ss lrve, when higher strch levels negtively ffected

10 10 these prmeters (Péres et l. 1996). It must e pointed out tht the mount of fish mel nd hydrolysed fish mel (CPSP G) used in ech diet provided enough n-3 polyunsturted ftty cid (round 1.5% of diet dry mtter) to sustin lrvl growth nd survivl (Chu et l., 2003). As expected, the growth rte of se ss lrve ws depressed in the group tht ws fed diet tht contined only 47% protein (SAP), nd in the group fed diet contining high concentrtion of hydrolyste (HHY). These effects were not due to low ingestion of the diet. In fct, it hd previously een shown tht optiml growth nd survivl in se ss re otined on diet tht contins 50-60% protein (Perés et l., 1996). Moreover, high levels of hydrolyste in the diet hve negtive effects on oth growth nd survivl, s demonstrted y Zmonino Infnte et l., (1997) nd Chu et l., (1999). Severl studies hd shown tht the inclusion of moderte proportion of protein hydrolyste improves lrvl development in severl species of fish such s crp (Crvlho et l., 1997), goldfish (Slminsk et l., 1991), se rem (Kolkovski nd Tndler, 2000), nd se ss (Chu et l., 1999). Nevertheless, growth ws efficient in the four groups, llowing vlule physiologicl studies to e otined. This study suggested tht, s erly s dy 25, trypsin ctivity in lrve is modulted t the trnsltionl level y the protein content of the diet. This result is originl for mrine fish lrve. Indeed, the fct tht Perés et l. (1996; 1998) filed to demonstrte such regultion in erly stges of se ss my hve een consequence of the poor growth of lrve in tht study. On dy 42, trnscriptionl control ws evidenced in trypsin regultion y the dietry protein content. Severl studies hve shown tht trypsin regultion is minly trnscriptionl in vertertes (Wicker et l., 1983; Brnnon 1990). Lhoste et l. (1994) demonstrted tht the nture of protein, csein, soyen or fish mel protein modultes trypsin mrna trnscription. Our study suggests tht the chin length of the protein my lso modulte trypsin trnscription

11 11 in se ss lrve. Additionl ssys erlier thn dy 25 nd fter dy 42 would llowed to confirm the switch from trnsltionl to trnscriptionl control of trypsin secretion, depending on the lrvl developmentl stge. Amylse expression decreses during lrvl development in severl crnivorous mrine fish: se ss (Zmonino Infnte nd Chu, 1994), se rem (Moyno et l., 1996), sole (Rieiro et l., 1999), red drum (Buchet et l., 2000), winter flounder (Dougls et l., 2000). The chnges in mylse expression were not necessrily dietry induced, nd could to correspond with the different feeding hits in the wild (Krogdhl nd Sundy, 1999). Such decrese in mylse cn e likened to the decrese in lctse which hs een descried in mmmls (Henning, 1987). High lctse ctivity chrcterizes the post-ntl stges in mmmls nd its decrese is ssocited with the mturtion of pncretic functions. Similrly, mylse ctivity cn e regrded s n indictor of pncres mturtion in mrine fish lrve (Chu nd Zmonino Infnte, 1994). In our study, in the three groups fed isoprotein diets, mylse expression (ctivity nd mrna levels) ws highest on dys 25 nd 42 in the HHYgroup tht exhiited the lowest rte of growth. This revels dely in the process of pncres mturtion. The high level of mylse ctivity in the SAP group is the result of oth dely in mturtion nd modultion of mylse y its sustrte, strch. Our results suggest tht the regultion of mylse expression is posttrnscriptionl in the erly stges (dy 25) nd ecomes trnscriptionl towrds the end of the lrvl period. We cn hypothesize tht mylse regultion is trnscriptionl in juvenile fish, s hs een reported in developing nd dult mmmls (Wicker et l., 1984). Previous studies conducted in fish suggested tht CCK expression is oth tissue-specific nd developmentlly regulted, s in other vertertes. In recent study only trce mounts of

12 12 CCK (0.2 fmol. ind -1 ) could e demonstrted in the gut of Atlntic hliut one week fter the onset of exogenous feeding, nd this represented only out 2% of the whole-ody content of CCK (Rojs-Grcí nd Rønnestd, 2002). However, fter further three weeks of development it incresed 100-fold to c 20 fmol. ind -1 ; representing 60% of the whole ody CCK content (Rojs-Grcí nd Rønnestd, 2002). In tht study the CCK content of the eviscerted ody, which proly derived primrily from sources in the centrl nervous system (CNS), ws kept lmost t constnt level of fmol. ind -1, regrdless of ody size during the period studied. Such finding cn e explined y more rpid increse in non- CCK-producing tissues (i.e. muscle) thn in the gut nd CNS. The cler llometric reltionship etween CCK content nd ody mss in our study (Fig. 2) indicte tht comprle reltionship lso occurs in developing se ss. Despite the lrge differences etween tretments in lrvl weight t the end of the experiment (Tle 2) nd the llometric reltionship of CCK per dry weight (Fig. 2), sttisticl evlution demonstrted significnt differences etween dietry groups, s reported here. We ssume tht the differences in CCK levels found in this study were minly those of the gstrointestinl trct, s the different dietry tretments were the only vrile to which the lrve were exposed. Similr ssumptions were pplied in n experiment on herring lrve y Koven et l. (2002) using the sme CCK ssy method s in the present study. However, it is evident tht in order to properly define the role of CCK in digestive function of lrvl fish, future studies should focus on seprting neurl nd gstrointestinl sources s well s differentiting etween the CCK stored in the producing cells (trnscription nd trnsltion) nd CCK relese.

13 13 Erlier studies on mmmls hve shown tht rise in dietry protein levels stimultes pncretic hypertrophy nd leds to incresed expression of pncretic enzymes (Green et l., 1986). These chnges in pncretic function re elieved to result from elevted plsm CCK concentrtions (Green et l., 1985). However, study of gene knockout mice hs shown tht CCK is not required s meditor for the dietry protein-induced increse in pncretic proteolytic enzymes (Lcourse et l., 1999). This supports erlier notions tht not ll effects on pncretic secretion cn e ttriuted to circulting CCK, since other hormones nd neurl stimultion might lso e involved (Liddle, 2000). In our study, the highest levels of trypsin ctivity in intestine were found in the three groups tht hd een fed high isoprotein diets. The CCK level in lrve did not follow the sme pttern, in tht it ws lrgely depressed in the group tht hd een fed diet incorporting protein hydrolyste (low level of ntive protein), compred to the other groups nd including the group fed diet strch-rich diet. This oservtion must e considered in conjunction with the depressed pncretic secretion of oth trypsin nd mylse in the group fed diet HHY (high protein hydrolyste nd low intct protein levels). Intct proteins re known to e the only stimulnts of CCK relese in rts (Liddle et l., 1986). Owyng (1994) showed tht csein hydrolyste, which induced trypsin ctivity, filed to stimulte CCK relese nd pncretic secretion in rt. We otined similr results in our study on fish lrve y using fish mel hydrolyste nd intct fish mel. These oservtions suggest tht dietry protein level nd chin length comined with intrluminl proteolytic ctivity my regulte the CCK level in fish lrve just s in rts. The high level of CCK ssocited with high pncretic enzyme secretion oserved in lrve fed the diet contining strch, my e due to the comined effects of low level of trypsin

14 14 (ecuse of low dietry protein concentrtion) in the intestinl lumen nd dely in lrvl development. The fct tht high level of CCK cn e found with low or high dietry protein level is comptile with existence of n indirect mechnism controlling CCK secretion in sess lrve. The presence of peptide tht regultes CCK relese nd medites pncretic enzyme secretion hs een demonstrted in some mmmls, including rts nd humns (Liddle, 1994). Under sl conditions, trypsin degrdes the CCK-relesing fctor. Food, prticulrly intct protein, which is sustrte for trypsin, my temporrily ind trypsin nd prevent degrdtion of the CCK-relesing fctor. Intct CCK-relesing fctor in turn stimultes the relese of CCK (Chey, 1993). The existence of such mechnism controlling CCK secretion in se ss lrve could explin some of our experimentl results. 5. Conclusion This study demonstrtes tht different levels of proteins nd inclusions of hydrolyste in the diets modulte trypsin expression nd in turn ffect CCK content of lrve. The study supports the hypothesis tht CCK is involved in protein digestion vi n indirect mechnism. We lso demonstrte tht high lrvl CCK content is not correlted with high rte of growth. Acknowledgements The uthors thnk M.M. Le Gll (enzyme ssys) nd P. Quzuguel (rering) for their excellent technicl support. RIA ssys were supported y Norwegin Reserch Council grnt /120. Dr. C.R. Rojs-Grcí is thnked for initil ssistnce with the CCK nlysis. The experiments conducted in this study comply with the current lws of the country in which the experiments were performed.

15 15 References Beccri, C., Diz, J.P., Connes, R., Chtin, B., Orgnogenesis of the exocrine pncres in the se ss, Dicentrrchus lrx L., rered extensively nd intensively. Aquculture 99, Bouhlic, M., Gudn, J., Histologicl study of the orgnogenesis of digestive system nd swim ldder of the Dover sole, Sole sole (Linneus 1758). Aquculture 102, Brdford, M.M., A rpid nd sensitive method for the quntittion of microgrm quntities of protein utilizing the principle of protein-dye inding. Anl. Biochem. 72, Brnnon, P.M., Adpttion of the exocrine pncres to diet. Annu. Rev. Nutr. 10, Buchet, V., Zmonino Infnte, J.L., Chu, C., Effect of lipid level in compound diet on the development of red drum (Scinops ocelltus) lrve. Aquculture 184, Chu, C.L., Zmonino Infnte, J.L., Erly wening of se ss (Dicentrrchus lrx) lrve with compound diet: effect on digestive enzymes. Comp. Biochem. Physiol. 109A, Chu, C., Zmonino Infnte, J.L., Quzuguel, P., Le Gll, M.M., Protein hydrolyste vs fish mel in compound diets for 10 dy old se ss Dicentrrchus lrx lrve. Aquculture 171, Chu, C., Zmonino Infnte, J.L., Bros V., Effect of dietry phospholipid level nd phospholipid: neutrl lipid vlue on the development of se ss (Dicentrrchus lrx) lrve fed compound diet. British J. Nutr. 90,

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18 18 Kurokw, T., Suzuki, T., Andoh, T., Development of cholecystokinin nd pncretic polypeptide endocrine systems during the lrvl stge of jpnese flounder, Prlichthys olivceus. Gen. Comp. Endocrinol. 120, Lcourse, K.A., Senerg, L.J., Gillespie, P.J., Rehfield, J.E., Sunders, T.L., Smuelsen, L.C., Pncretic function in CCK deficient mice: dpttion to dietry protein does not require CCK. Am. J. Physiol. 276, G1302-G1309. Lhoste, E.F., Fiszlewicz, M., Gueugneu, A.M., Trnchnt, T., Corring, T., Erly dpttion of pncres to protein-enriched diet: role of cholecystokinin nd gstrinrelesing peptide. Pncres 9, Liddle, R.A., Regultion of cholecystokinin gene expression in rt intestine. Ann. N.Y. Acd. Sci. 713, Liddle, R.A., Regultion of cholecystokinin secretion y intrluminl relesing fctors. Am. J. Physiol. 269, G319-G327. Liddle, R.A., Cholecystokinin cells. Annu. Rev. Physiol. 59, Liddle, R.A., Regultion of cholecystokinin secretion in humns. J. Gsteroenterol. 35, Liddle, R.A., Green, G.M., Conrd, C.K., Willims, J.A., Proteins ut not mino cids, crohydrtes, or fts stimulte cholecystokinin secretion in the rt. Am. J. Physiol. 251, G243-G248. Métis, P., Bieth, J., Détermintion de l α-mylse pr une microtechnique. Ann. Biol. Clin. 26, Moyno, F.J., Diz, M., Alrcon, F.J., Srsquete, M.C., Chrcteriztion of digestive enzyme ctivity during lrvl development of gilthed se rem (Sprus urt). Fish Physiol. Biochem. 15,

19 19 Owyng, C., Negtive feedck control of exocrine pncretic secretion: role of cholecystokinin nd cholinergic pthwy. J. Nutr. 124, 1321S-1326S. Péres, A., Chu, C., Zmonino Infnte, J.L., Le Gll, M.M., Quzuguel, P., Amylse nd trypsin response to dietry crohydrte nd protein level depends on the developmentl stge in se ss (Dicentrrchus lrx) lrve. Fish Physiol. Biochem. 15, Péres, A., Zmonino Infnte, J.L., Chu, C., Dietry regultion of ctivities nd mrna levels of trypsin nd mylse in se ss (Dicentrrchus lrx) lrve. Fish Physiol. Biochem. 19, Rehfeld, J.F., Accurte mesurement of cholecystokinin in plsm. Clin. Chem. 44(5), Rieiro, L., Zmonino Infnte, J.L., Chu, C., Dinis, M.T., Development of digestive enzymes in lrve of Sole seneglensis, Kup. Aquculture 179, Rojs-Grcí, C.R., Rønnestd, I., Cholecystokinin nd tryptic ctivity in the gut of developing Atlntic hliut (Hippoglossus hippoglossus): evidence for prticiption in the regultion of protein digestion. J. Fish Biol. 61, Rojs-Grcí, C.R., Rønnestd, I., Ueerschäer, B., Comined sensitive nlyticl methods for cholecystokinin levels nd tryptic ctivity in individul fish lrve. J. Exp. Mrine Biol. Ecol. 265, Singer, M., Neurohormonl control of pncretic enzyme secretion in nimls. In: Go, V.L.W. et l. (Eds.), The Pncres: Biology, pthoiology, nd disese, 2 nd edition. Rven Press, New York, pp Sölch, J.P., Arnold, G.J., Multiplex reverse trnscription polymerse chin rection comined with temperture grdient gel electrophoresis s tool for the normlized quntifiction of intrinsic fctor mrna. Electrophoresis 17,

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21 21 Tle 1. Composition of the experimentl diets DIETS Ingredients 1 (in %) NAP LHY HHY SAP Fish mel Hydrolyzed fish mel Precooked Potto strch Cod liver oil Soy lecithin Vitmin Mixture 2 Minerl Mixture 3 Betine Proximl composition Proteins (Nx6.25) Lipids Ash % dry mtter Dietry ingredients were commercilly otined. Fish mel, Hydrolyzed fish mel (CPSP G, Concentré de Protéines Solules de Poisson) nd cod liver oil were from L Lorientise (Lorient, Frnce). The soy lecithin ws from Ets Louis Frnçois (St Mur des Fossés, Frnce). The pottoe precooked strch (Nutrlys) ws from Roquette (Lille, Frnce). 2 Per kg of vitmin mix: retinyl cette 1 g; choleclciferol 2.5 mg; ll-rc- α-tocopherol cette 10 g; mendione 1 g; thimin 1 g; rioflvine 0.4 g; D- clcium pntothente 2 g; pyridoxine HCl 0.3 g; cynocolmin 1 g; nicin 1 g; choline chloride 200 g; scoric cid 20 g; folic cid 0.1 g; iotine 1 g; meso-inositol 30 g. 3 Per kg of minerl mix: KCl 90 g; KI 40 mg; CHPO4. 2H 2 O 500 g; NCl 40 g; CuSO 4. 5H 2 O 3 g; ZnSO 4. 7H 2 O 4 g; CoSO 4. 7H 2 O 20 mg; FeSO 4. 7H 2 O 20 g; MnSO 4. H 2 O 3 g; CCO g; MgSO 4. 7H 2 O 124 g; NF 1 g.

22 22 Tle 2. Growth nd survivl rte t dy 42 of se ss lrve fed the four experimentl diets. Mens ± S.D. (n=4) with different letters in sme row re significntly different (P<0.05). DIETS NAP LHY HHY SAP Lrvl growth Initil weight (mg) 1.0 ± 0.92 Finl weight (mg) 21.6 ± ± ± ± 2.40 Survivl rte (%) 44.9 ± ± ± ± 5.31 Tle 3. Specific ctivity nd RNA levels of trypsin nd mylse in homogentes of pncres segment of se ss lrve fed the different experimentl diets. Mens ± S.D. (n=4 for enzyme ctivity; n=3 for RNA rtio) with different letters in sme row re significntly different (P<0.05). Enzyme Trypsin DIETS NAP LHY HHY SAP Activity in mu/mg protein 64 ± ± ± ± 4.8 Dy 25 Dy ± ± ± ± 8.3 RNA rtio Dy ± ± ± ± 0.09 Dy ± ± ± ± 0.06 Amylse Activity in U/mg protein 1.7 ± ± ± ± 0.60 Dy 25 Dy ± ± ± ± 0.50 RNA rtio Dy ± ± ± ± 0.07 Dy ± 0.18c 1.2 ± 0.07c 1.7 ± ± 0.08

23 23 Trypsin in intestinl segment munit/mg prot NAP LHY HHY SAP 0 D25 D42 Figure 1. Specific ctivity of trypsin ssyed in intestinl segment of se ss lrve fed the four experimentl diets. Mens ± S.D. (n=4) with different superscript letters for sme dy re significntly different (P<0.05). 80 CCK weight specific content (fmol/mg, dm) ,0 1,0 2,0 3,0 4,0 5,0 6,0 7,0 8,0 9,0 Fish dry mss (mg, dm) Figure 2. Reltionship etween mount of CCK nd dry mss of whole se ss lrve.

24 24 A B CCK in fmol/lrve NAP LHY HHY SAP D25 D42 80,0 70,0 60,0 50,0 40,0 30,0 20,0 10,0 - D25 D42 c C D Trypsin Secretion in % D25 c D42 Amylse Secretion in % D25 D42 Figure 3. CCK in whole ody lrve (3A) or relted to dry weight lrve (3B). Secretion level [% of enzyme ssyed in the intestinl segment (mu/segment) divided y enzyme ssyed in pncretic plus intestinl segment] of trypsin (3C) nd mylse (3D) in se ss lrve. Mens ± S.D. (n=4) with different letters for sme dy re significntly different (P<0.05).