Ontogeny of the digestive tract of thick lipped grey mullet (Chelon labrosus) larvae reared in mesocosms

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1 Plese note tht this is n uthor-produced PDF of n rticle ccepted for publiction following peer review. The definitive publisher-uthenticted version is vilble on the publisher Web site Aquculture July 28, Volume 279, Issues 1-4, Pges Elsevier B.V. All rights reserved. Archimer Archive Institutionnelle de l Ifremer Ontogeny of the digestive trct of thick lipped grey mullet (Chelon lbrosus) lrve rered in mesocosms Dor Zouiten, Ines Ben Khemis, *, Rouf Besbes nd Chntl Chu b INSTM (Institut Ntionl des Sciences et Technologies de l Mer), Lbortoire Aquculture, BP 59, 5 Monstir, Tunisi b Unité Mixte INRA-Ifremer de Nutrition des Poissons, BP-7 Ifremer, Plouzné, Frnce *: Corresponding uthor : Ben Khemis I., Tel.: ; fx: , emil ddress : ines.benkhmis@instm.rnrt.tn Abstrct: This work describes the ontogeny of the digestive trct in thick lipped grey mullet (Chelon lbrosus) lrve rered until dy 36 post-htching with the semi-extensive technology in mesocosms. Diet ws constituted by live preys, rotifers, Artemi nd wild zooplnkton, then compound diet ws dded from dy 2 (p. h.). Liner growth, weight growth nd digestive enzymes specific ctivities were studied during lrvl ontogeny. Pncretic enzymes (trypsin nd mylse) nd intestinl enzymes (leucinelnine peptidse Leu-l, minopeptidse N AN nd lkline phosphtse AP ) were ssyed in lrve smpled throughout the rering tril to evlute gstrointestinl mturtion long the development. The trypsin specific ctivities were very high during the first two weeks nd then declined s observed in mrine fish species. A following increse in trypsin specific ctivity from dy 2 ws ttributed notbly to ingestion of prticle compound diet. In contrst to the pttern generlly described in fish lrve, mylse specific ctivity showed continuous increse. This could be ttributed to the fct tht C. lbrosus is n omnivorous species nd suggests tht the fish might be ble to use efficiently diets contining higher levels of strch or other crbohydrtes since the end of lrvl development. Reltive expression of intestinl brush border membrne enzymes (AP nd AN) nd cytosolic enzyme (Leu-l), showed n brupt increse of both AP/leu-l nd AN/leu-l rtios t dy 8 (p. h.), indicting tht mturtion of intestinl trct in C. lbrosus lrve is prticulrly precocious. It is ssumed tht lrve of C. lbrosus might support erly co-feeding nd wening strtegies, which could resonbly be initited since mouth opening. Keywords: Chelon lbrosus; Mesocosm; Fish lrve; Intestinl enzymes; Pncretic enzymes 1

2 1. Introduction Mesocosm fish lrvl rering ppers n interesting nd simple technology for mss production of thick lipped grey mullet Chelon lbrosus juveniles, trget species for quculture (Crossetti nd Ctudell, 1995) nd impounded wter seeding progrms (Ben Khemis et l., 26). This htchery technique is bsed on n environment, which offers nturl food prticles during the first criticl feeding period of the fish lrve (Ns et l. 1991; Divnch nd Kentouri, 2). It hs been used for lrvl rering of mny species with different ecologicl chrcteristics (Ginnkourou, 1995; Nehr et l., 1996; Ben Khemis, 1997; Berg, 1997; Ppndroulkis et l., 24, 25; Zouiten et l., 24; Ziss et l., 26). These studies pointed out tht mesocosms provide n dequte environment for lrvl development. In ddition, uthors documented better qulity of mesocosm rered lrve nd their similrity to wild concerning morphologicl chrcteristics (Koumoundouros et l., 1997; Boglione et l., 21). The low technology pproch is considered solution for species of moderte or low mrket vlue (Lee nd Ostrowski, 21) or smll producers (Shields, 21). In mesocosm rering technique, food from exogenous origin is dded in order to void over grzing of the nturl food chin by the top predtor of the system, i.e. the lrve (Divnch nd Kentouri, 2). This dded food includes both rered live preys (Georgls et l., 27) nd compound diet (Ben Khemis et l., 26) nd is essentil for improving fish growth nd survivl. The formultion of compound diet dpted to the lrvl requirement of fish species necessittes the understnding of the development of digestive system of this species. Development of digestive enzyme hs been widely studied in mny crnivorous fish species: se brem (Clzd et l., 1998); winter flounder Pleuronectes mericnus (Dougls et l., 1999); se bss (Chu nd Zmbonino-infnte, 21; Zmbonino-Infnte nd Chu, 21), Atlntic cod Gdus morhu nd Atlntic hlibut (Kvåle et l, 27) or omnivorous fish such s crp Cyprinus crpio (Escffre et l.1997). Some studies reported the effect of the rering condition or wening on the development of digestive trct nd enzymes (Chu et l., 1998; Hmz et l., 27). Other works hve shown tht diet composition ffects positively or negtively lrvl enzymtic development (Zmbonino-Infnte nd Chu, 1994; Buchet et l., 2; Tovr et l., 22; Svoie et l. 26). The mturtion process of digestive enzyme cn be enhnced, stopped or delyed depending on composition of diet (Zmbonino-Infnte nd Chu, 21). However, very few dt concerning digestive enzyme development in thick lipped grey mullet re reported in literture. Nevertheless, growth chrcteristics nd first dt on phosphtse lkline ctivity suggested specilly erly development of intestine function in thick lipped grey mullet (Ben Khemis et l. 26). The im of the present work ws to study the development of digestive system of the species, in order to provide dt on lrve digestive trits during ontogeny, to verify the hypothesis of precocious digestive trct mturtion nd to determine whether digestion ppers lredy oriented towrd omnivorous feeding since erly stges. Trypsin ( protese) nd mylse ( glucosidse) were both ssyed to study the functionl development of exocrine pncres. For intestinl digestion, leucine lnine peptidse, cytosolic peptidse nd minopeptidse N nd lkline phosphtse, two brush border membrne enzymes, were ssyed to study enterocytes mturtion. These enzymes were ssyed in Chelon lbrosus lrve smpled throughout n experimentl mesocosm rering tril. 2. Mterils nd methods 2.1. Broodstock spwning nd lrvl trnsfer The study ws relized t the mrine quculture center of INSTM Institut Ntionl des Sciences et Technologies de l Mer t Monstir (Tunisi). Thick lipped grey mullet fertilized eggs were obtined from cptive broodstocks, kept since severl yers in 1 m 3 concrete tnks t the mrine quculture center of INSTM. Two femles (3. nd 2.7 kg) received HCG nd LHRH following the protocol described in Besbes et l, (22). The injected femles nd four running mles (men weight of 1.6 kg) were plced in spwning tnk nd spontneous spwns were obtined. The pooled spwn produced 782 eggs nd the fertiliztion rte ws 68%. The eggs were trnsferred in n incubtion unit with flow through of filtered nd UV sterilized se wter (T=18.6; ph=8.45; S=37.7). Incubtion lsted 5 dys nd htching rte ws 82%. On dy 1 (p. h.), 2

3 lrve were distributed into three mesocosms enclosures t rering density of 1.5 lrve/liter (3 lrve per enclosure of 2 m 3 ) Experimentl nd rering conditions Three cylindricl concrete tnks of 2 m 3 cpcity coted with PVC liner were used s mesocosms. These enclosures were protected by polyester-glss fibre greenhouse nd shded by individul blck griculturl curtins fixed horizontlly t 2 m over the wter surfce. Initil filling nd subsequent renewl during rering used nturl sewter pumped in the djcent Lgun of Khniss nd filtered on 36 μm mesh. Before lrvl distribution, mturtion period of 7 dys during which wter ws mintined stgnnt, ws pplied for plnkton development (Divnch nd Kentouri, 2). Wter qulity ws monitored dily for temperture, ph nd dissolved oxygen. The rte of wter renewl ws mintined t 1% per dy during the first week of rering nd then it ws progressively incresed to rech 4% on dy 15 (p. h.) nd 1% on dy 35 (p. h.). It ws djusted ccording to dissolved oxygen contents of wter to gurntee t lest 5 mg l -1. Nturl nd uncontrolled plnkton blooms were let to develop within the mesocosms but in bsence of ny wter fertiliztion to void excessive phytoplnkton development (Ben Khemis et l., 26) nd relted lrvl deth (Ben Khemis, 1997). Clening of enclosures by siphoning sedimented wstes ws initited on dy 1 (p. h.) nd ws performed, ech other dy before wening. After wening, clening ws performed dily to void wter pollution. Siphoned wstes were filtered nd checked for ded fish or excess of uneten food. Totl mmoni nitrogen, which is the min hrmful product of fish metbolism nd should be mintined below.5 ppm, generlly does not represent mjor problem in the lrvl unit due to scrce lrvl biomss (Moretti et l., 1999; 25). With wter renewl, bsence of fertiliztion, siphoning of sedimented wstes nd development of phytoplnkton, totl mmoni nitrogen did not exceed.2 ppm in mesocosm enclosures. The dy of filling, wter qulity prmeters were T=18.6 C, S=37.5 nd ph=8.2. Wter temperture remined constnt for the 7 dys before the stocking of fish lrve. After lrvl distribution, it decresed grdully to 15±1 C until dy 11 (p. h.) but remined higher thn in se, where nturl reproduction tkes plce in the wild. Then temperture incresed progressively to 19.9±.6 C nd remined lmost constnt until the end of experimentl tril. Aertion ws provided in ech enclosure by two ir diffusers positioned on the bottom ner the tnk wll nd dimetriclly opposed. Nturl plnkton blooms were let to develop within the mesocosms. Phytoplnkton ws not quntified but densities of wild zooplnkton, corresponding to endogenous preys, were estimted dily from 1 l of wter siphoned within enclosures nd filtrted on 3µm mesh to finl volume of 5 ml. Three sub-smples of 1 ml from the concentrted smple were then observed in toto under binoculr stereomicroscope for counting the zooplnktons Lrvl nd juvenile feeding nd rering The live preys included both wild zooplnkton nturlly developing in the enclosures, nd rered Brchionus nd Artemi. Rered preys were enriched, ccording to the instructions provided by the mnufcturer, with DHA-Protein Selco (INVE) nd DC-Super Selco (INVE) respectively. Adjunction of rered preys ws performed once dily in the morning fter zooplnkton smpling. Until beginning of wening (dy 2 p. h.), the density of potentil preys, considered s the sum of endogenous nd dded zooplnkton, ws mintined over.3 individul ml 1. The wild zooplnkton ws dominted by copepods, minly nuplii nd copepodite stges, nd wild rotifers s Synchet. Dily men preys densities of the enclosures re given in Fig. 1. Consumption of newly htched Artemi type AF (INVE) ws tested since dy 1 (p.h) but distribution in rering enclosures ws initited on dy 12 (p. h.) fter hving observed its effective cpture by the lrve. Between dy 16 nd dy 2, smll size Artemi (type AF - INVE) ws progressively shifted to one dy enriched lrge size Artemi (type EG - INVE). The delivery of the commercil formulted diet Replce II from Rich SA (1-3 µm) begn on dy 2 (p. h.), fter development of intestinl dult mode of digestion ccording to our preliminry study (Ben Khemis et l., 26). Formulted feed proximl composition ws 58% proteins, 18% lipids, 8% crbohydrtes. The complete wening onto rtificil diet ws chieved on dy 33 (p. h.) fter co-feeding period of 16 dys, including three dys of drstic reduction of Artemi rtions. Dily rtions of formulted feed were 1, 5, 7 nd 2 g per enclosure from dy 2 to dy 23, from dy 24 to dy 28, from dy 29 to dy 31 nd from dy 32 to dy 3

4 36, respectively. These rtions, bsed on previous results nd observtion of both behvior nd feed ingestion, were subdivided into 12 to 2 frctions nd distributed mnully. During co-feeding, t lest four mels were given prior ny live preys distribution (Ben Khemis et l., 26) Lrvl smpling nd nlysis Lrve were lwys smpled before morning food distribution. For growth study, 15 lrve were smpled in ech enclosure dily from dy 1 to dy 1 nd then on dy 14, 2, 28 nd 36. They were nesthetized with ice-cold sewter, then fixed with glutrldhyde (2.5% in phosphte buffered solution ph 7.4) nd kept refrigerted until length nd weight mesurement. Photogrphs of the fish were tken using digitl cmer (Nikon Coolpix 45) mounted on trin-oculr stereomicroscope (Hund Wetzlr) nd length mesurements were crried out using imge nlysis softwre ImgeJ An object micrometer ws photogrphed with ech set of photos to void errors due to the uto-focus of the cmer. Drined weights of fixed lrve were mesured immeditely fter photogrphing. Lrve were weighed individully, except for specimens of less thn 5mg (i.e. lrve less then 2 dys old) for which pooled weights were tken. For enzymtic ssy, lrve were smpled on dy, 4, 8, 14, 2, 28 nd 36. Smples corresponded to 12, 7, 6, 4, 3, 2, 15 lrve collected from ech enclosure, respectively. They were immeditely stored t - 8 C pending ssy. Before homogenizing in ice cold distilled wter, lrve were vortexed in 5 µl ice cold distilled wter during 3 sec to obtin relesed enzyme (superntnt S1). This superntnt contined the secreted pncretic enzymes, i.e. trypsin nd mylse (M et l., 25). The lrve were then homogenized in 1 to 2 ml ice cold distilled wter, depending of the weight of the smple, with homogenizer (Polytron, PT-MR 21) during 3 sec then centrifuged t 33 g for 3min. This superntnt (S2), ws used to nlyze unrelesed pncretic enzymes (trypsin nd mylse) nd intestinl enzymes (lkline phosphtse [AP], minopeptidse N [AN] nd leucine-lnine peptidse [Leu-l]). Amylse nd trypsin ctivities were ssyed ccording to Métis nd Bieth (1968) nd Holm et l. (1988), respectively. The brush border membrne enzymes, lkline phosphtse nd mminopeptidse N were ssyed ccording to Bessey et l. (1946) nd Mroux et l. (1973), respectively. Assy of the cytosolic leucine-lnine peptidse ws performed using the method of Nicholson nd Kim (1975). Activities were mesured s µmoles of substrte hydrolysed min/mg protein, t 37 C for AP, Leu-l nd AN, nd t 25 C for trypsin (Zmbonino-Infnte nd Chu, 1994). Amylse ctivity represented the equivlent enzyme ctivity required for hydrolyzing 1 mg of strch in 3 min t 37 C (Zmbonino-Infnte nd Chu, 1994). Enzyme ctivities were expressed s segmentl ctivity (sum of S1 nd S2 mu or U/lrve) nd specific ctivity (mu or U/mg protein). Protein ws determined by the Brdford procedure (Brdford, 1976). The result re given s mens ± S.D (n=3). The vrince homogeneity of the dt ws checked using Levene s test. Rtios of segmentl enzymes ctivities were rcsin(x 1/2 ) trnsformed. Results were compred by nlysis of vrince (ANOVA) followed by the Tukey s test when differences were found t α=.5 (Zr, 1999). Sttistics were performed using Sttistic 5.5 (SttSoft, Inc.). 3. Results The lrve mesured 3.41±.48 mm in totl length t dy 1 (p. h.) nd 3.84±.31 mm t dy 5 (p. h.) when mouth opened. During ll the rering period, no significnt differences of sizes or weights were observed between the lrve of the different enclosures. The results were hence nlyzed globlly fter pooling. The liner growth of the lrve could be divided into two distinct phses. From htching to dy 14 (p. h.) lrvl growths ws remrkbly slow. At tht stge, the lrve mesured 4.5±.5 mm. Afterwrd, growth ccelerted shrply nd the lrve reched 11.2±1.72 mm on dy 29 (p. h.) nd 14.12± 2.67 mm on dy 36 (p. h.). Initil body weight of lrve ws.62±.2 mg. It reched 28.53± 9.15 mg t the end of the rering tril. Both liner growth (Fig. 2A) nd weight growth (Fig. 2B) presented similr ptterns. Segmentl ctivities of pncretic (Fig. 3A) nd intestinl (Fig. 3B) enzymes, which reflect enzyme content per lrv, showed n increse comprble to lrve growth. Trypsin nd mylse ctivities were detected from 4

5 htching, both in superntnt (S1) nd in pncretic tissues (S2). We choose to present specific ctivity of secreted pncretic enzymes s this frction (in S1) corresponds to the ctive enzyme in the intestinl lumen. Trypsin specific ctivity incresed t mouth opening nd remined high the following dys (Fig. 4A). It decresed shrply t dy 14 (p. h.) nd fterwrds it showed progressive increse until dy 28 (p. h.) nd remined t high level until the end of the experiment. Amylse specific ctivity showed regulr increse from htching to dy 2 (p. h.) nd reched plteu (Fig. 4B). Specific ctivities of Leu-l peptidse (tble 1) decresed progressively between htching nd dy 8 (p.h.) while specific ctivities of minopeptidse N nd phosphtse lkline incresed with lrvl ge from mouth opening until dy 14 (p. h). Then, minopeptidse specific ctivity mintined t sme level while lkline phosphtse specific ctivity decresed. Both rtios of intestinl enzymes AN/Leu-l nd AP/Leu-l showed shrp increse t dy 8 (p. h.) (Fig 5). Afterwrds, AN/Leu-l rtio vried slightly until the end of the experiment while AP/Leu-l rtio decresed progressively. 4. Discussion Growth of Chelon lbrosus lrve (size nd weight) in the present work exhibited similr pttern of chnges compred to the previous descriptions of Ctudell et l. (1988) nd Boglione et l. (1992) or our erlier work (Ben Khemis et l., 26). It seems hence tht the lower initil rering tempertures did not ffect lrve development in mjor wy. It comes out tht the two first weeks of life of C. lbrosus lrve re chrcterized by remrkbly slow growth. We previously suggested tht this extended low initil growth is chrcteristic of erly lrvl development of mullets nd proposed tht it could be due to lloction of vilble energy nd building mterils in priority to physiologicl chnges (mong which digestive functions) rther thn in size growth. The study of digestive development is consistent with this hypothesis. During the present experiment, lrve received live prey, until dy 32 (p. h.). This diet cn be considered s nturl food llowing proper development in mullet like in other mrine fish lrve species, s pointed out by M et l. (25) in the yellow croker (Pseudoscien croce). The ontogenetic chnges of the intestinl trct of thick lipped grey mullet observed in this work should represent theoriclly the norml development pttern, i.e. the pttern which should be observed in the wild, t the sme temperture, when lrve cn et d libitum. It hs been demonstrted tht digestive trct nd digestion process undergo mjor developmentl chnges during the first weeks of life in fish (Wlford nd Lm, 1993; Srsquete et l., 1995; Zmbonino-Infnte nd Chu, 1994, 21). Pncres secretion function constitutes the first step of mturtion process of digestive function, nd the second is the onset of brush border membrne enzymes in the intestine (M et l., 25). Lrve of Chelon lbrosus exhibited high trypsin ctivities during endogenous feeding (before mouth opening) nd the first feeding dys, indicting lrge potentil to synthesize enzymes, s generlly observed in fish lrve (Chu nd Zmbonino-Infnte, 21). The observed decline in trypsin specific ctivity which reched the minimum t 14 (p. h.) should be ttributed to lrve growth, nd thus to n increse of body protein. In fct, specific ctivity is the rtio ctivity per mg protein nd does not reflect lowering in digestive cpcity (M et l., 25), s demonstrted by the continuous increse of segmentl trypsin ctivity. So, the initil pttern of trypsin enzyme ctivities during thick lipped grey mullet development ws similr to tht lredy described in other species, sebss, sole nd red drum (Zmbonino-Infnte nd Chu, 21). The increse of trypsin specific ctivity observed from dy 2 onwrds could be ttributed to two fctors. In one hnd, growth shrply incresed from dy 2 nd trypsin hs been shown to be growth indictor in fish lrve (Rungrungsk- Torrissen et l., 26). On the other hnd, enriched Artemi nd compound diet were introduced since dy 16 nd dy 2 (p. h.), respectively. Hence, the food quntity considerbly incresed in mesocosms. It ws demonstrted in fish lrve tht trypsin ctivity responds to n increse in dietry rtions (Zmbonino-infnte et l., 1996; Pedersen et l., 23). Furthermore, the compound diet incorportes high protein level, 58%, nd trypsin ctivity should be relted to dietry protein concentrtion s reported by Péres et l. (1998) in se bss lrve. Amylse specific ctivity hs been shown to be high during young lrvl stges nd generlly decrese during the development of the lrve (Chu nd Zmbonino-Infnte, 21). In this study, mylse specific ctivity 5

6 showed continuous increse during the first 2 dys of lrvl development nd ws mintined t high level onwrds. This continuous increse nd elevted plteu should not be induced by n ugmenttion of dietry crbohydrtes s observed by M et l (25) in yellow croker lrve. Indeed, the compound diet ws introduced on dy 2 (p. h.), i.e. t the end of mylse specific ctivity incresing period. In ddition, it contined 8% crbohydrtes, which is not different of crbohydrte content of copepods nd Artemi (M et l., 25). Thus, this cpcity to synthesize mylse t developed lrvl stges cn be ttributed to C. lbrosus digestive specificity. Comprtive studies of digestive enzymes in fish with different nutritionl hbits hve demonstrted tht mylse ctivity is greter in herbivorous nd omnivorous fish thn in crnivorous fish (Hidlgo et l., 1999; Fernández et l., (21). Informtion on digestive enzymes of young mullets is scrce (Ds et l., 1987; Brmn et l., 25). But it is known tht dult thick lipped grey mullets feed minly on benthic ditoms, epiphytic lge, smll invertebrtes nd detritus (Ben-Tuvi, 1986; Crossetti nd Ctudell, 1994). The cpcity of synthesizing mylse nd thus to feed on vegetl resources seems to be expressed since erly lrvl development stges in C. lbrosus. The existence of well developed mylse synthesis cpcity my be n interesting feture from the perspective of feed formultion (Fernández et l., 21). The young thick lipped grey mullets would be ble to use efficiently diets contining high levels of strch or other similr low cost mylolitic energetic compounds. The utiliztion of crbohydrtes cn help chieve the development of diets with minimized cost s it is n inexpensive source of energy (Lzo et l., 27). The intestinl enzymes ctivities ssyed in C. lbrosus lrve, exhibited inverse evolutions between cytosolic nd brush border membrne enzymes. Leucine lnine peptidse specific ctivity decresed from htching until minimum reched t dy 8 (p.h.) while both phosphtse lkline nd minopeptidse N incresed. This pttern is typicl of intestinl mturtion, indicting tht brush border enzymes relyed cytosolic enzyme for digestion s described in Zmbonino-Infnte nd Chu (21). The rtio of brush border membrne enzyme ctivity to cytosolic enzyme ctivity is considered s n indictor of the development of intestinl digestion (Zmbonino-Infnte nd Chu (21). Indeed, mturtion of intestine epithelium of lrve is initited few dys fter htching with the differentition of the brush border membrne (BBM) of enterocytes (Vu, 1983). Then, the mturtion process is chrcterized by shrp increse in BBM enzymes (Kvåle et l., 27) ssocited in some species with coexisting decrese in cytosolic enzymes (Zmbonino-Infnte nd Chu, 21). The mturtion process of enterocytes hs been extensively described in severl fish species such s common crp Cyprinus cprio (Escffre et l., 1997), se brem Sprus urt (Clzd et l., 1998), red drum Scienops ocelltus (Buchet et l., 2), yellow croker Pseudoscien croce (M et l., 25; Mi et l., 25). A proper chievement of this process determines lrvl survivl ccording to Chu nd Zmbonino-Infnte (1995). In se bss lrve, the mturtion of intestine, which chrcterizes the dult mode of intestinl digestion, ends pproximtely in the fourth week of post-htching development (Chu nd Zmbonino-Infnte, 1995). In cold wter species such s cod (Gdus morhu) nd hlibut (Hippoglossus hippoglossus) the mturtion occurs lter, t pproximtely 4 to 5 dys post first feeding (Kvåle et l., 27) while in subtropicl species such s red drum (Scinops ocelltus), it occurs round dy 18 (Buchet et l., 2). In our study, both AP/leu l nd AN/leu l rtios incresed shrply t dy 8 (p. h.) indicting tht mturtion of intestinl trct of thick lipped grey mullet is prticulrly precocious. These results suggest tht thick lipped grey mullet lrve could be wened t very erly stges, just few dys fter mouth opening. In fct, the intestinl mturtion ws ssocited to the cquisition of cpcity of mrine fish lrve to digest compound diet, i.e to be wened successfully; yet good wening results depend on diet tht fulfils the lrvl requirement nd tkes into ccount the digestive fetures (Kvåle et l., 27). After intestinl mturtion, AP/leu l rtio showed significnt decreses in young C. lbrosus, contrrily to the pttern generlly observed in fish lrve. It could be ttributed to erly cquisition of digestive fetures of the species feeding regime. Germn et l. (24) observed decrese in lkline phosphtse ctivities in two herbivorous pricklebck species (Cebidichthys violceus nd Xiphister mucosus) s the fish shifted from crnivore diet towrd n lgl diet during ontogeny. Furthermore, these uthors showed tht digestive enzyme ctivity is geneticlly progrmmed to mtch ontogenetic shifts in diet. 6

7 5. Conclusion This study presents the first dt on digestive enzymes quntifiction in C. lbrosus lrve. The results point out tht the cpcity of synthesizing mylse nd thus to feed efficiently on vegetl sources seems to be expressed since erly lrvl development stges in C. lbrosus. Hence diet with higher levels of glycolytic compounds could be introduced into lrvl feeding sequence. The results lso indicte tht mturtion of intestinl trct is prticulrly precocious, suggesting tht these lrve could be wened t very erly stges, just few dys fter mouth opening. Using lst genertions of high performnce microdiets, we would resonbly suggest pplying co-feeding strtegies since mouth opening. Acknowledgements This work ws supported by the reserch project entitled TENMIA funded by the Tunisin Ministry of Scientific Reserch, Technology nd Development of Competences nd the project of coopertion between INSTM nd Ifremer, supported by the Embssy of Frnce in Tunisi. Prticulr thnks re ddressed to Dr Amel Medhioub nd the htchery stff of the INSTM s well s Dr José Zmbonino-Infnte, Mrie Mdeleine le Gll, for their help nd technicl ssistnce. 7

8 References Brmn, U. K., Jn, S.N., Grg, S.K., Bhtngr, A.; Arsu, A.R.T. 25. Effect of inlnd wter slinity on growth, feed conversion efficiency nd intestinl enzyme ctivity in growing grey mullet, Mugil cephlus (Linn.): Field nd lbortory studies. Aqucult. Int., 13(3), Ben Khemis, I Elevges lrvires de poissons méditerrnéens: optimistion de l production en mésocosme et diversifiction des espèces. Thèse de doctort, Université de droit, d économie et des sciences d'aix-mrseille III, 186 pp. Ben Khemis, I., Zouiten D., Besbes, R., Kmoun, F., 26. Lrvl rering nd wening of thick lipped grey mullet (Chelon lbrosus) in mesocosm with semi-extensive technology. Aquculture, 259, Ben-Tuvi, A., Mugilide. In: Fishes of the North-estern Atlntic nd Mediterrnen. P.J.P. Whitehed, M.-L. Buchot, J.-C. Hureu, J. Nielsen nd E. Tortonese (eds.), Volume 3, UNESCO, Pris. : Besbes, R., Fuvel, C., Guerbej, H., Benseddik Besbes, A., El Ouer, A., Kriem, M.M., El Abed, A., 22. Contribution à l étude de l mturtion et de l ponte en cptivité du mulet lippu Chelon lbrosus (Cuvier, 1829). Résultts préliminires de pontes pr stimultion hormonle. Actes des 5èmes Journées Tunisiennes des Sciences de l Mer, December 22, Aïn Drhm, Tunisi. Bulletin de l'instm, n spécil (7), Bessey, O.A., Lowry, O.H., Brock, M.J., Rpid coloric method for determintion of lkline phosphtse in five cubic millimeters of serum. J. Biol. Chem. 164, Boglione, C., Bertolini, B., Russiello, M., Ctudell, S., Embryonic nd lrvl development of the thicklipped mullet (Chelon lbrosus) under controlled reproduction conditions. Aquculture, 11, Boglione, C., Gglirdi, F., Scrdi, M., Ctudell, S., 21. Skeletl descriptors nd qulity ssessment in lrve nd post-lrve of wild-cught nd htchery-rered gilthed se brem (Sprus urt L. 1758). Aquculture, 192, Brdford, M.M., A rpid nd sensitive method for the quntittion of microgrm quntities of protein utilizing the principle of protein dye binding. Anl. Biochem. 72, Buchet, V., Zmbonino Infnte, J.L., Chu, C.L., 2. Effect of lipid level in compound diet on the development of red drum (Scienops ocelltus) lrve. Aquculture, 184, Chu, C., Zmbonino Infnte, J.L., Mturtion of the pncretic nd intestinl digestive functions in se bss (Dicentrrchus lbrx): effect of wening with different protein sources. Fish Physiol. Biochem. 14, Chu, C., Zmbonino Infnte, J.L., Escffre, A.M., Bergot, P., Kushik, S., Preliminry results on se bss Dicentrrchus lbrx lrve rering with compound diet from first feeding, comprison with crp Cyprinus crpio lrve. Aquculture 169, 1 7. Chu, C., Zmbonino Infnte, J.L., 21. Substitution of live food by formulted diets in mrine fish lrve. Aquculture 2, Clzd, A., Medin, A., Gonzáles de Cnles, M.L., Fine structure of the intestine development in cultured se brem lrve. J. Fish Biol., 53, Ctudell, S., Crosetti, D., Mss, F., Rmpcci, M., The propgtion of juvenile mullet (Chelon lbrosus) in erthen ponds. Aquculture, 68, Crosetti, D., Ctudell, S., The mullets. In: Nsh, C.E., Novotny, A.J., (Eds.), World Animl Science - Production of qutic nimls (fishes-c8). Elsevier, Amsterdm, pp Ds, K.M., Ghosh, A., Ghosh, A., Studies on the comprtive ctivity of some digestive enzymes in fry nd dult of mullet Liz prsi (Hm.). J. Aqucult. Tropics, 2(1), Divnch, P., Kentouri, M., 2. Htchery techniques for specific diversifiction in Mediterrnen finfish lrviculture. Recent Advnces in Mediterrnen Aquculture Finfish Species Diversifiction. Proceedings of the TECAM Seminr, My 1999, Zrgoz, Spin. Chiers Option Méditerrnéennes, vol. 47, pp Dougls, S.E., Gwlick, A., Mndl, S., Gllnt, J.W., Ontogeny of the stomch in winter flounder: chrcteristion nd expression of the pepsinogen nd proton pump genes nd determintion of pepsin ctivity. J. Fish Biol., 55 (5), Escffre, A.M., Zmbonino Infnte, J.L., Chu, C.L., Mmbrini, M., Bergot, P., Kushik, S., Nutritionl vlue of soy protein concentrte for lrve of common crp (Cyprinus crpio) bsed on growth performnce nd digestive enzyme ctivities. Aquculture, 153,

9 Fernández, I., Moyno, F.J., Dìz, M., Mrtìnez, T., 21. Chrcteriztion of -mylse ctivity in five species of Mediterrnen sprid fishes (Spride, Teleostei). J. Exp. Mr. Bio. Ecol., 262, Georgls, V., Mlvsi, S., Frnzoi, P., Torricelli, P, 27. Swimming ctivity nd feeding behviour of lrvl Europen se bss (Dicentrrchus lbrx L): Effects of ontogeny nd incresing food density. Aquculture, 264, Germn, D.P., Horn, M.H., Gwlick, A., 24. Pricklebck Fishes (Teleostei: Sticheide): Ontogenetic, Dietry, nd Phylogenetic Effects. Physiologicl nd Biochemicl Zoology, 77(5), Ginnkourou, A Réseux trophiques plnctoniques utilisbles pour l'élevge lrvire de l durde (Sprus urt, 1758). Thèse de Doctort, Université des Sciences et Techniques du Lnguedoc, Montpellier, 24 pp. Hmz, N., Mhetli, M., Kestemont, P., 27. Effects of wening ge nd diets on ontogeny of digestive ctivities nd structures of pikeperch (Snder lucioperc) lrve. Fish Physiol Biochem, Fish Physiol. nd Biochem., 33: Hidlgo, M.C., Ure, E., Snz, A., Comprtive study of digestive enzymes in fish with different nutritionl hbits. Proteolytic nd mylse ctivities. Aquculture, 17, Holm, H., Hnssen, L.E., Krogdhl, A., Florholmen, J., High nd low inhibitor soyben mels ffect humn duodenl proteinse ctivity differently: in vivo comprison with bovine serum lbumin. J. Nutr. 118, Koumoundouros, G., Gglirdi, F., Divnch, P., Boglione, C., Ctudell, S., Kentouri, M Norml nd bnorml osteologicl development of cudl fin in Sprus urt L. fry. Aquculture, 149, Kvåle, A., Mngor-Jensen, A., Moren, M., Espe, M., Hmre, K., 27. Development nd chrcteristion of some intestinl enzymes in Atlntic cod (Gdus morhu L.) nd Atlntic hlibut (Hippoglossus hippoglossus L.) lrve. Aquculture 264 : Lzo, J.P., Mendoz, R., Holt, G.J., Aguiler, C., Arnold, C.R., 27. Chrcteriztion of digestive enzymes during lrvl development of red drum (Scienops ocelltus). Aquculture, 265, Lee, C.S., Ostrowski, A.C., 21. Current sttus of mrine finfish lrviculture in the United Sttes. Aquculture, 2, M, H., Chu, C., Zmbonino, J., Yu, H., Dun, Q., Le Gll, M.M., Mi, K., 25. Activities of selected digestive enzymes during lrvl development of lrge yellow croker (Pseudoscien croce). Aquculture, 245, Mi, K.,Yu, H., M, H., Dun, Q., Gisbert, E., Zmbonino Infnte, J.L., Chu, C., 25. A histologicl study on the development of the digestive system of Pseudoscien croce lrve nd juveniles. J. Fish Biol., 67, Mroux, S., Louvrd, D., Brtti, J., The minopeptidse from hog-intestinl brush border. Biochim. Biophys. Act 321, Métis, P., Bieth, J., Determintion de l -mylse pr une microtechnique. Ann. Biol. Clin. 26, Moretti, A., Pedini Fernndez-Crido, M., Cittolin, G., Guidstri, R., Mnul on Htchery Production of Sebss nd Gilthed Sebrem. Volume 1. Rome, FAO: 194 p. Moretti, A., Pedini Fernndez-Crido, M., Vetillrt, R., 25. Mnul on Htchery Production of Sebss nd Gilthed Sebrem. Volume 2. Rome, FAO: 152 p. Ns, K. E., Vn Der Meeren, T., Aksnes, D. L., Plnkton succession nd responses to mnipultions in mrine bsin for lrvl fish rering. Mr. Ecol. Prog. Ser., 74, Nehr, O., Blncheton, J.P., Alliot, E., Development of n intensive culture system for se bss (Dicentrrchus lbrx) lrve in enclosures. Aquculture, 142, Nicholson, J.A., Kim, Y.S., A one-step l-mino cid oxidse ssy for intestinl peptide hydrolse ctivity. Anl. Biochem. 63, Ppndroulkis, N., Kentouri, M., Mingot, E., Divnch, P., 24. Mesocosm: relible technology for lrvl rering of Diplodus puntzzo nd Diplodus srgus srgus. Aqucult. Int., 12, Ppndroulkis, N., Mylons, C.C., Mingot, E., Divnch, P., 25. First results of greter mberjck (Seriol dumerili) lrvl rering in mesocosm. Aquculture, 25, Pedersen, B.H., Ueberschär, B., Kurokw, T., 23. Digestive response nd rtes of growth in preleptocephlus lrve of the Jpnese eel Anguill jponic rered on rtificil diets. Aquculture, 215,

10 Péres, A., Zmbonino Infnte, J.L., Chu, C.L., Dietry regultion of ctivities nd mrna levels of trypsin nd mylse in se bss (Dicentrrchus lbrx) lrve. Fish Physiol. Biochem., 19, Rungrungsk-Torrissen, K., Moss, R., Andresen, L.H., Berg, A., Wgboe, R., 26. Different expressions of trypsin nd chymotrypsin in reltion to growth in Atlntic slmon (Slmo slr L.). Fish Physiol. Biochem, 32(1),7-23. Srsquete, M.C., Polo, A., Yúfer, M., Histology nd histochemistry of the development of the digestive system of lrvl gilthed sebrem Sprus urt L. Aquculture, 13, Svoie, A., Le Frnçois, N.R., Chu, C., Blier, P.U., Andressen, I., 26. Do protein hydrolystes improve survivl nd growth of newly-htched spotted wolffish (Anrhichs minor), non-metmorphic quculture fish species? Aquculture, 261, Shields, R. J., 21. Lrviculture of mrine finfish in Europe. Aquculture, 2, Tovr, D., Zmbonino, J., Chu, C., Gtesoupe, F.J., Vzquez-Jurez, R., Lésel, R., 22. Effect of live yest incorportion in compound diet on enzyme ctivity in se bss (Dicentrrchus lbrx) lrve. Aquculture, 24, Vu, T.T., Etude histoenzymologique des ctivités protésiques dns le tube digestif des lrves et des dultes de br, Dicentrrchus lbrx (L). Aquculture, 32, Wlford, J., Lm, T.J., 1993 : Development of digestive trct nd proteolytic enzyme ctivity in sebss (Ltes clcrifer) lrve nd juveniles. Aquculture, 19, Ziss, M.M., Ppdkis, I.E., Mingot, E., Divnch, P., Mylons, C., 26. Ontogeny of the digestive trct in shi drum (Umbrin cirros L.) rered using the mesocosm lrvl rering system. Aquculture, 26, Zmbonino Infnte, J.L., Chu, C., Development nd response to diet chnge of some digestive enzymes in se bss (Dicentrrchus lbrx) lrve. Fish Physiol. Biochem., 12 (5), Zmbonino Infnte, J.L., Chu, C.L., Péres, A., Quzuguel, P., Le Gll, M.M., 1996; Se bss (Dicentrrchus lbrx) lrve fed different Artemi rtions: growth, pncres enzymtic response nd development of digestive functions. Aquculture, 139, Zmbonino Infnte, J.L., Chu, C., 21. Ontogeny of gstrointestinl trct of mrine fish lrve. Comp. Biochem. Physiol. C, 13, Zr, J. H Biosttisticl Anlysis (4th Edition). Prentice Hll, Englewood Cliffs, 663 pp. Zouiten, D., Msmoudi, A.S., El Abed, A., Hell, A.N., Ben khemis, I., 24. Co-feeding nd erly wening of the Europen se bss (Dicentrrcus lbrx) under semi-extensive conditions in "mesocosms" in Tunisin winter geoclimtic context. Biologi Mrin Mediterrne, 11(2),

11 Figures 1,4 1,2 Enriched Artemi Newly htched AF Artemi Enriched Brchionus plictilis Wild zooplnkton Preys / ml 1,,8,6,4,2, Lrvl ge (dys p. h. ) Fig. 1: Men dily estimtion of endogenous nd exogenous live prey densities in rering enclosures. Arrow indictes beginning of compound diet delivery. (A) 2 (B) 4 Lrvl size (mm) Lrvl weight (mg) Lrvl ge (dys p. h.) Lrvl ge (dys p. h.) Fig. 2: Growth of Chelon lbrosus lrve in mesocosm. Dt re given in men ± SD nd re expressed in mm for totl body length (A) nd in mg for weight (B). 11

12 (A) 75 2,5 Trypsin Segmentl ctivity (mu) Amylse 2, 1,5 1,,5 Segmentl ctivity (U) , (B) Segmentl ctivity (mu) Phosphtse lkline Leu-l peptidse Aminopeptidse N Segmentl ctivity (U) Lrvl ge (dys p.h.) Fig. 3: Segmentl pncretic (A) nd intestinl (B) enzyme ctivities in C. lbrosus lrve. Dt re given in men ± SD (n=3). They re expressed in mu for trypsin, minopeptidse N nd phosphtse lkline nd in U for mylse nd leucine-lnine peptidse. 12

13 (A) Secreted trypsin (S1) 6 mu/mg protein 45 3 b b 15 c (B) Secreted mylse (S1) 1 U/mg protein b b c d Lrvl ge (dys p.h.) Fig. 4: Specific ctivities of secreted trypsin (A) nd mylse (B) during lrvl development of C. lbrosus lrve. Dt re given in men ± S.D (n=3). Different letters indicte significntly different vlues (α=.5) ccording to Tukey s test. 13

14 (A) Segmentl (AN / Leu-Al) x AN/Leu-Al b b bc b c d (B) Segmentl (AP/Leu-Al) x AP/Leu-Al b c d e d d Lrvl ge (dys p.h.) Fig. 5: Rtios of intestinl enzymes, AN/Leu-l (A) nd AP/Leu-l (B) during lrvl development of C. lbrosus. Dt re given in men ± S.D (n=3). Different letters indicte significntly different vlues (α=.5) ccording to Tukey s test. 14

15 Tbles Cytosolic enzyme Brush border enzymes Age Leu-l peptidse Aminopeptidse N Alkline phosphtse (dy p.h.) (U/mg protein) (mu/mg protein) (mu/mg protein) 55,56 ±5,65 (d) 1,3 ±3,55 (d) 26,11 ±3,74 (d) 4 42,3 ±1,7 (e) 7,53 ±1,68 (d) 25,18 ±1,9 (d) 8 33,7 ±2,89 (f) 16,5 ±1,62 (c) 112,69 ±17,55 (b) 14 8,51 ±3,97 (b) 24,77 ±1,69 (b) 14,88 ±5,28 () 2 83,28 ±2,3 (b) 28,87 ±1,1 (b) 16,25 ±5,22 (b) 28 65,16 ±1,71 (c) 28,7 ±1,26 (b) 51,24 ±6,33 (c) 36 13,56 ±1,47 () 31,63 ±2,25 () 66,88 ±3,55 (c) Tble 1: Specific ctivities of intestinl enzymes ssyed on the lrve homogentes of C. lbrosus. Dt re given in men ± S.D (n=3). Different letters indicte significntly different vlues (α=.5) ccording to Tukey s test. 15