Activity of digestive enzymes in yolk-sac larvae of Atlantic halibut ž Hippoglossus hippoglossus /: indication of readiness for first feeding

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1 Ž. Aquculture Activity of digestive enzymes in yolk-sc lrve of Atlntic hlibut ž Hippoglossus hippoglossus /: indiction of rediness for first feeding Ann Gwlick,), Brigitte Prent, Michel H. Horn b, Neil Ross, Ingegjerd Opstd c, Ole J. Torrissen c Institute for Mrine Biosciences, Ntionl Reserch Council Cnd, 1411 Oxford Street, Hlifx, NoÕ Scoti, Cnd B3H 3Z1 b Deprtment of Biologicl Science, Cliforni Stte UniÕersity, Fullerton, CA , USA c Institute of Mrine Reserch, AusteÕoll Aquculture Reserch Sttion, N-5392 Storebø, Norwy Received 24 Mrch 1999; received in revised form 23 September 1999; ccepted 24 September 1999 Abstrct The problem of determining when lrve should be offered food is prticulrly difficult in species such s Atlntic hlibut tht hs long yolk-sc period Ž degree dys, dd.. In order to help determine t wht ge Atlntic hlibut lrve re ble to digest food, we compred the ctivities of key digestive enzymes in four yolk-sc stges t n ge intervl tht hs been recommended for initition of feeding, i.e., dd. We tested the hypothesis tht digestive enzyme ctivities rech highest levels ner the end of this ge intervl. Activities of trypsin, mylse, lipse nd lkline phosphtse were determined spectrophotometriclly in whole yolk-sc lrve t 161, 179, 230, nd 276 dd. The ctivities of the sme enzymes were mesured in metmorphic lrve Ž 660 dd. nd in their Artemi prey to provide reference levels from fully developed digestive system nd to estimte the importnce of exogenous enzymes for Atlntic hlibut lrve. Our results showed significnt Ž P differences in ctivities of ll four digestive enzymes mong the yolk-sc stges with generl pttern of increse from 161 to 276 dd. Trypsin ctivities reched their highest vlues t 230 dd, wheres those of lipse nd lkline phosphtse peked t 276 dd. Amylse ctivities were detected only in the 230 nd 276 dd stges, t sttisticlly indistinguishble levels. Bsed on percentge comprisons, specific ctivities of trypsin nd mylse in whole 276-dd lrve were only 12% nd 2%, respectively, of those mesured in the digestive system of metmorphic lrve, wheres specific ctivities of lipse nd ) Corresponding uthor. Deprtment of Biologicl Science, Cliforni Stte University t Fullerton, Fullerton, CA , USA. Tel.: q ; fx: q ; e-mil: gwlick@fullerton.edu r00r$ - see front mtter q 2000 Elsevier Science B.V. All rights reserved. Ž. PII: S

2 304 ( ) A. Gwlick et l.raquculture lkline phosphtse in 276-dd lrve were more thn 50% of those determined for metmorphic lrve. The clculted contribution of enzyme ctivities derived from Artemi prey to the reltively high levels of enzyme ctivity in the digestive system of metmorphic lrve ws less thn 10% for ll enzymes except mylse, for which the contribution ws estimted to be more thn 50%. The results of this study support our hypothesis tht the highest digestive enzyme ctivities in yolk-sc lrve re reched by dd, i.e., ner the end of the ge intervl recommended for first feeding. The observed pttern of enzyme ctivities suggests tht feeding of Atlntic hlibut lrve should be initited fter 230 dd, but not lter thn 276 dd to void the thret of strvtion. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Alkline phosphtse; Amylse; Artemi; Lipse; Trypsin 1. Introduction The time period during which mrine fish lrve in nture begin to feed is criticl phse in their lives becuse it ffects their survivl, growth nd development Žsee Snderson nd Kupferberg, Lrve switch to n exogenous food supply whenever they re developed enough to ingest nd digest food nd bsorb the nutrients. In the commercil rering of mrine fish lrve, this trnsition from endogenous Ž yolk. to exogenous feeding is often period of high mortlity. One of the min chllenges of rering lrvl fishes in cptivity is to find wys to minimize this mortlity. Meeting this chllenge requires knowledge bout when the lrve of the species in question re cpble of exogenous feeding nd thus when they should be offered food. In turn, knowing when to initite feeding must be bsed on sound understnding of the structurl nd functionl development of the digestive system. In other words, the lrve must be cpble of digesting the food consumed if they re to survive nd grow. The chllenge of rering mrine fish lrve is compounded becuse species vry in the time of first exogenous feeding s they exhust their yolk supply nd begin to rely incresingly on externl food sources. The lrve of some common species, such s Atlntic cod, Europen sebss, plice, turbot nd winter flounder Ž Pleuronectes mericnus., htch from smll eggs Ž 1 2 mm. with limited mount of yolk nd hve short Ž 2 6 dys. yolk-sc period Ž Jobling, 1995; Rønnestd et l., The current rering prctice for these species is to begin feeding the lrve when the mouth hs opened but before the yolk-sc is completely resorbed Ž Wtnbe nd Kiron, In other species, such s Atlntic hlibut, the lrve htch from lrger eggs Ž mm. with greter yolk reserve nd experience longer, temperture-dependent yolk-sc period, which lsts for 280 Ž Olsen et l., to 320 Ž Lein nd Holmefjord, degree dys Ž dd s wter temperture, 8C, =ge, dys post-htch.. Clerly, the problem of deciding when to offer food to lrve with such n extended yolk-sc period is different nd rgubly more difficult thn for lrve with much shorter yolk-sc period. This problem is prticulrly cute in Atlntic hlibut for which lmost nothing is known bout the size t first feeding in the wild. Severl pproches hve been used to determine when feeding should be initited in Atlntic hlibut lrve, but hve resulted in discrepncies for the recommended ge for first feeding. Studies bsed on histomorphology of the digestive system ŽKjørsvik nd

3 ( ) A. Gwlick et l.raquculture Reiersen, 1992., morphology nd behvior Ž Blxter et l., 1983; Pittmn et l., 1990b., lgl uptke Ž Reitn et l., 1994., reltive protein synthesis Ž RNArDNA rtio. ŽPittmn et l., 1990; Skiftesvik et l., nd on respirtion, nitrogen nd energy metbolism Ž Finn et l., in developing yolk-sc lrve recommend tht feeding should be strted t ge dd, i.e., when bout 50 30% of the yolk-sc remins. Feeding t this erly ge would be dvntgeous in commercil sense Ž Pittmn, becuse the dded nutrient intke would ccelerte growth nd shorten the time of production. The results, however, of feeding experiments show tht Atlntic hlibut lrve should be offered food t lter ge intervl, either dd Ž Lein nd Holmefjord, 1992., dd Ž Reitn et l., or dd Ž Hrboe nd Mngor-Jensen, Although the current prctice is to begin feeding lrve round dd ŽGr et l., 1998., the initil food uptke remins low nd represents the min bottleneck in intensive rering Ž Shields et l., Recent reserch hs demonstrted tht initil feeding success cn be incresed by postponing first feeding to dd ŽHrboe nd Mngor-Jensen, Although the feeding t this lter ge my be dvntgeous in n economic sense Ž Hrboe nd Mngor-Jensen, 1998., erlier work by Hjelmelnd et l. Ž recommended tht, bsed on trypsinogenrtrypsin content, feeding should be initited no lter thn 280 dd in order to void strvtion. Solving the dilemm of time of first feeding in Atlntic hlibut requires reserch on the poorly known ontogenetic sequence of digestive enzyme ctivity in order to determine t wht ge the lrve re ble to digest nd bsorb exogenous nutrients. The present study ws designed to compre the ctivities of key digestive enzymes in four yolk-sc stges of Atlntic hlibut lrve representing lrgely the ge intervl tht hs been recommended for initition of feeding, i.e., dd. We tested the hypothesis tht digestive enzyme ctivities rech highest levels ner the end of this ge intervl. If digestive enzyme ctivity is good indictor of lrvl digestive cpcity, then the time of highest ctivity should indicte when the lrve hve become physiologiclly redy to process exogenous food. We mesured the levels of digestive enzyme ctivity in metmorphic lrve Ž 660 dd. to provide reference levels from more highly developed digestive system while recognizing tht metmorphic lrve my exhibit higher levels of digestive enzyme ctivity thn the yolk-sc lrve in prt becuse enzymes of their prey my contribute to totl enzyme ctivity. In order to estimte the importnce of exogenous enzymes for Atlntic hlibut lrve, we clculted the reltive contribution of enzyme ctivities derived from Artemi prey to those mesured in the digestive system of metmorphic lrve. 2. Mterils nd methods 2.1. Rering of lrõe Yolk-sc lrve of Atlntic hlibut, Hippoglossus hippoglossus, were obtined nd rered following the stndrd protocol used t the Austevoll Aquculture Reserch Sttion of the Institute of Mrine Reserch, Storebø, Norwy Ž Hrboe et l., 1994,b..

4 306 ( ) A. Gwlick et l.raquculture Eggs were stripped from one femle nd fertilized with milt from one mle. After fertiliztion, eggs were incubted t 68C in 250-l conicl tnks with slow upwelling inflow. One dy before htching, eggs were moved to one 5-m 3 upwelling silo supplied with snd-filtered sewter Ž t 6 78C nd kept in drkness. Wter flow ws 2 lrmin for the first 2 weeks nd 4 lrmin therefter. Lrve htched fter 84 dd. By 161 dd, survivl ws 87% nd remined high during the second hlf of the yolk-sc period, but only 5% of lrve survived by 276 dd becuse of brekdown of the min wter pump. An dditionl btch of yolk-sc lrve ws obtined from commercil fltfish htchery Ž Austevoll Mrin Yngelprodusjon., nd rered in circulr fiberglss tnks Ž Hrboe et l., supplied with green sewter Ž Næss et l., t 128C nd n inflow of 1 lrmin. Feeding ws initited on 265 dd with enriched brine shrimp Ž Artemi. nuplii Ž McEvoy et l., distributed three times dy t rte of 1000 nupliirlrdy. Lrve were lso given nturl zooplnkton between 6 nd 20 dys of feeding s recommended by Næss et l. Ž From 650 dd, the feeding rte nd wter inflow were incresed progressively to 6000 nupliirlrdy nd 6 lrmin, respectively Smpling of lrõe Yolk-sc lrve were collected t 161, 179, 230 nd 276 dd Ži.e., 26, 29, 37 nd 44 dys post-htch, dph. nd euthnized individully with n overdose of metomidte hydrochloride Ž 0.01 grl, Wildlife Phrmceuticls, Fort Collins, CO, USA.. Lrve were plced on glss surfce kept on ice nd exmined under dissecting microscope to eliminte individuls with bnormlly formed jws. One hundred normlly formed lrve were grouped by tens, dried of residul wter with pper toweling, nd then frozen in liquid nitrogen. At 161, 179, 230 nd 276 dd, men wet Ž vs. dry, 48 h t 608C. body msses of individul, unfrozen lrve Ž n s 10. were 6.4 Ž 0.5., 6.6 Ž 0.6., 5.8 Ž 0.8. nd 5.3 Ž 0.5. mg, respectively, with SE Ž - 10%. in ech cse. Ten metmorphic lrve with norml pigmenttion, symmetric bodies nd completely migrted left eye were smpled t 660 dd Ž 78 dph. fter 34 dys of feeding. Lrve were dried with pper toweling, weighed individully Žmen wet body mss of 56.0"9.0 mg. nd dissected on glss surfce kept on ice nd 10 whole digestive systems Ž including liver nd pncres. contining Artemi were frozen. Photogrphs of five dditionl metmorphic lrve from the sme group were tken in order to provide n estimte of the number of Artemi nuplii in digestive system. Smples of live Artemi nuplii were collected on 80-mm mesh sieve nd trnsferred to five replicte cryo-vils with c. 14,000 Artemi nuplii per vil before freezing in liquid nitrogen. All smples were stored t y808c until processed Enzyme ssys Frozen whole yolk-sc lrve Ž n s 5 pooled smples of 10 lrve. nd digestive systems of metmorphic lrve Ž n s 5. were prtilly thwed, weighed nd homogenized on ice in five volumes of 0.2 M NCl Ž wrv. using motorized teflon pestle. Frozen smples of Artemi were homogenized on ice in two volumes of 0.2 M NCl. This sline solution ws chosen becuse it is nturlly present in the lumen of the

5 ( ) A. Gwlick et l.raquculture digestive system of mrine fishes. The suspensions were centrifuged t 12,000 = g for 5 min nd the superntnts plced on ice nd used immeditely for spectrophotometric determintion of enzyme ctivities nd soluble protein content following the protocols optimized on dult guts nd described below. All ssys were crried out in triplicte t room temperture Ž 238C. using Thermomx microplte spectrophotometer ŽMoleculr Devices, Sunnyvle, CA, USA.. Blnks were used to ccount for non-enzymtic hydrolysis of substrtes. The concentrtions given correspond to those in the finl incubtion mediums. Trypsin Ž E.C ctivity ws ssyed following Erlnger et l. Ž Homogentes were incubted with 1 mm BAPNA ŽN--benzoyl-L-rginine p-nitronilide hydrochloride, Boehringer Mnnheim, ct. no in 25 mm mmonium bicrbonte buffer, ph 7.8, nd the increse in bsorbnce ws mesured t 405 nm for 30 min. Amylse Ž E.C ws mesured ccording to the Somogyi Nelson procedure Ž Somogyi, Strch substrte ws prepred by boiling 1% soluble strch ŽSigm, S2630. in 0.8 M sodium citrte buffer, ph 7.0, for 5 min. Smples were incubted with 0.5% strch in 0.2 M sodium citrte t ph 7.0, ph considered optiml for mylse in dult Atlntic hlibut Ž Glss et l., The incubtion ws stopped fter 6 h by dding 0.2 volumes of 1 M NOH nd two volumes of the first Somogyi Nelson regent. Reducing sugrs were determined by mesuring the chnges in bsorbnce t 650 nm. Lipse Ž nonspecific, E.C ctivity ws mesured ccording to modified method of Albro et l. Ž Homogentes were incubted with 0.4 mm p-nitrophenyl myristte Ž Sigm, N2502. in 24 mm mmonium bicrbonte, ph 7.8, contining 0.5% Triton X-100 s n emulsifying gent. The chnge in bsorbnce ws mesured t 405 nm for 30 min. Alkline phosphtse Ž E.C ws ssyed following Wlter nd Schutt Ž Homogentes were incubted with 4 mm p-nitrophenyl phosphte Ž Sigm, N6750. in 55 mm mmonium bicrbonte buffer with 0.6 mm MgCl 2, ph 7.8. The increse in bsorbnce ws mesured continuously for 30 min t 405 nm. Soluble protein content in homogentes of whole lrve nd digestive systems ws determined by the method of Brdford Ž 1976., using bovine gmm-globulin ŽBioRd, Mississug, Ontrio, Cnd. s stndrd. Concentrtions of the ssy products were determined experimentlly bsed on stndrd curve of bsorbnce vs. product concentrtion using the sme volumes s in the ssys. All ctivities determined for the digestive enzymes were within the dynmic rnge of the respective ssys. Enzyme ctivity ws expressed s specific ctivity Ž murmg protein. or tissue ctivity Ž murmg tissue. nd represented nnomoles of product liberted during 1 min of hydrolysis per milligrm of protein or milligrm of whole lrve Ž dd. or digestive systems Ž 660 dd Sttisticl nlyses Men vlues of enzyme ctivities nd soluble protein contents were compred mong Ž. the yolk-sc stges with one-wy ANOVA Minitb, Stte College, PA, USA. Results

6 308 ( ) A. Gwlick et l.raquculture tht were significntly different were nlyzed further by Fisher multiple-comprison test. An level of 0.05 ws estblished priori for ll sttisticl tests. 3. Results Significnt differences in specific nd tissue ctivities of ll four digestive enzymes were found mong the yolk-sc stges with generl pttern of increse from 161 to 276 dd Ž Tble 1.. Trypsin ctivities reched their highest vlues t 230 dd, wheres those of lipse nd lkline phosphtse peked t 276 dd. Amylse ctivities were detected only in the 230 nd 276 dd stges, t sttisticlly indistinguishble levels. When expressed in murmg protein, the ctivities of lipse nd lkline phosphtse decresed significntly from the 161- to the 179-dd stge, prlleling n increse in soluble protein content. Bsed on percentge comprisons, specific ctivities of trypsin nd mylse in whole 276-dd lrve were only 12% nd 2%, respectively, of those mesured in the digestive systems of metmorphic lrve, wheres specific ctivities of lipse nd lkline phosphtse in 276-dd lrve were more thn 50% of those determined for metmorphic lrve. Men specific ctivities of the digestive enzymes in Artemi nuplii re given in Tble 2. At lest 200 nuplii Ž rnge were found in the digestive system of Tble 1 Activities of digestive enzymes nd content of soluble proteins in whole lrve t four yolk-sc stges U Ž dd. nd in the whole digestive system of metmorphic lrve Ž 660 dd. of Atlntic hlibut EnzymerProtein Lrve Ž dd. Yolk-sc Metmorphic Specific ctiõities ( murmg protein) Trypsin 3.4"0.1 b 9.3"0.8 d 16.7"0.7 c 11.6" "18.6 b b Amylse 0.0" " " " "401.0 c b d Lipse 15.7" " " " "2.8 Alkline phosphtse b 34.2" "1.1 b 38.5"1.1 c 61.2" "7.2 Tissue ctiõities ( murmg tissue) Trypsin 0.1"0.0 c 0.3"0.0 d 0.4"0.0 b 0.2" "1.8 b b Amylse 0.0" " " " "20.2 b Lipse 0.3" " " " "0.2 Alkline phosphtse 0.6" "0.1 b 0.8"0.0 c 1.0" "0.5 Soluble protein contents ( m grmg tissue) 16.8"1.2 c 27.1"1.1 b 21.2" " "2.8 U Mens"1SEŽns5 pooled smples of 10 yolk-sc lrve or ns5 digestive systems of metmorphic lrve. were compred mong yolk-sc stges only. ŽSpecific ctivities, Fs85.3, trypsin; 20.5, mylse; 75.8, lipse; 58.2, lkline phosphtse. Tissue ctivities, Fs82.2, trypsin; 11.4, mylse; 32.7, lipse; 29.0, lkline phosphtse. Soluble protein contents, Fs24.1. P nd df s19 in ll cses, except, df s17 for mylse.. Loction nd number of significntly different mens re designted by different superscript letters Ž Fisher test..

7 ( ) A. Gwlick et l.raquculture Tble 2 Specific ctivities Ž murmg protein. of digestive enzymes mesured in Artemi prey nd their percentge contribution to the totl specific ctivity of these enzymes clculted for the whole digestive system of metmorphic lrve of Atlntic hlibut contining 200 Artemi nuplii per lrv. See Section 2.2 for dditionl detils Enzyme Activity in Clculted ctivity % Contribution of Artemi 200 Artemi Digestive system Artemi enzymes Trypsin 52.6" Amylse 5,449" Lipse 6.3" Alkline phosphtse 68.8" Mens"1 SE Žns3 pooled smples of c. 14,000 Artemi nuplii or ns3 digestive systems of metmorphic lrve.. metmorphic lrve. The percentge contribution of 200 such nuplii to totl enzymtic ctivity in the digestive system of these lrve ws less thn 10% for ll enzymes except mylse for which the contribution ws estimted to be more thn 50%. 4. Discussion The results of this study support our hypothesis tht the highest digestive enzyme ctivities re reched ner the end of the ge intervl Ž dd. recommended for first feeding in Atlntic hlibut lrve. We found, however, differences in the time nd mount of chnge in ctivity mong the four enzymes studied, nd these my reflect the extended sequence of functionl development in the digestive system of fish, such s Atlntic hlibut, with long yolk-sc period. The pttern of trypsin ctivity showing pek t the 230-dd yolk-sc stge followed by decrese t 276 dd suggests tht lrve of Atlntic hlibut should be fed in this intervl of development. The pek in trypsin ctivity indictes incresed functionlity of the pncres Ž Segner et l., 1994; Kurokw nd Suzuki, 1996., wheres the subsequent decline my be sign of pncretic tissue degenertion from strvtion ŽKjørsvik et l., 1991; Ueberschr, 1993; Tnk et l., This pttern ppers to be consistent with incresed trypsinogenrtrypsin content t dd nd its decrese by 280 dd reported for Atlntic hlibut Ž Hjelmelnd et l., In other mrine fish lrve, ll with short yolk-sc periods, first feeding correltes with incresed trypsinogenrtrypsin content Ž Hjelmelnd et l., 1984; Pedersen et l., 1987; Kurokw nd Suzuki, nd incresed trypsin ctivity ŽLuff nd Hofer, 1984; Ueberschr, 1993; Zmbonino Infnte nd Chu, 1994; Oozeki nd Biley, For Atlntic hlibut lrve, the pttern ppers to be similr, but the timefrme is necessrily extended becuse of the long yolk resorption period. The pttern of mylse ctivity, showing no detectble levels t 161 nd 179 dd nd reltively low levels t 230 nd 276 dd compred to other vlues reported for mrine fish lrve t first feeding ŽZmbonino Infnte nd Chu, 1994, 1999; Oozeki nd Biley, 1995., suggests limited bility of Atlntic hlibut yolk-sc lrve to digest

8 310 ( ) A. Gwlick et l.raquculture crbohydrtes. This limited bility my explin the low efficiency Ž 1 5%. with which Atlntic hlibut ssimilte microlge throughout the yolk-sc period ŽReitn et l., The inefficient use of crbohydrtes by Atlntic hlibut nd other crnivorous mrine fishes tht inhbit cold wters is not surprising given tht mylse ctivity is known Ž Munill-Morn nd Sborido-Rey, to be low t 58C, which is one of the stndrd rering tempertures Ž 5 68C. of hlibut yolk-sc lrve. Lrve of mrine fish, including Atlntic hlibut Ž see below., tht hve been eting zooplnkton, however, my exhibit higher mylse ctivity. In wlleye pollock Ž Thergr chlcogrmm., Oozeki nd Biley Ž showed tht most of this incresed ctivity origintes from the zooplnkton prey, whose mylses exhibit lower temperture optim thn those of fish Ž Myzud, The pttern of continuous increse in lipse ctivity through the yolk-sc period in Atlntic hlibut suggests tht the lrve re prepred to feed on lipid-rich zooplnkton by 276 dd. This high lipse ctivity in the lter stges of yolk-sc resorption coincides with the period of incresed use of lipids for energy by Atlntic hlibut lrve ŽRinuzzo et l., 1992; Finn et l., 1995; Rønnestd et l., nd grees with recent studies on lipses in other mrine fish lrve ŽOzkizilcik et l., 1996; Zmbonino Infnte nd Chu, Further reserch is required to verify tht lipolytic cpcities in Atlntic hlibut lrve re limited by low bile slt production s suggested by Rønnestd et l. Ž The pttern of continuous increse in ctivity of lkline phosphtse through the yolk-sc period in Atlntic hlibut suggests tht high bsorptive cpcities of the intestine re ttined by 276 dd. Alkline phosphtse ctivity hs been ssocited with bsorption of extrcellulr nutrients Ž see Zuev et l., nd its increse used s n indictor of the onset of bsorptive function in the intestinl epithelium of fish lrve ŽCousin et l., 1987; Zmbonino Infnte nd Chu, 1994; Gwlick et l., 1995; Bglole et l., A decrese in lkline phosphtse ctivity usully ccompnies strvtion Ž Cousin et l., or the feeding of n indequte diet ŽChu nd Zmbonino Infnte, 1994; Gwlick et l., An increse in ctivity of this enzyme, however, lso hs been observed in mlnourished sebss lrve Ž Zmbonino Infnte nd Chu, Whether the high levels of lkline phosphtse in the unfed hlibut lrve in our study signled higher bsorptive cpcities or strvtion remins to be determined by histologicl exmintion of the intestine in 276 dd lrve. Our results suggest tht mylse but not trypsin, lipse or lkline phosphtse from Artemi prey contributes importntly to digestive cpcity in metmorphic lrve of Atlntic hlibut. The high mylse ctivity we recorded in these 660 dd lrve prllels the ctivity level of this enzyme in metmorphic sebss lrve fed Artemi ŽZmbonino Infnte nd Chu, Artemi nuplii re herbivores nd re expected to hve high mylse levels in order to digest the crbohydrtes found in the microlge they re fed in culture Ž Semin et l., Thus, not surprisingly, the minimum clculted contribution of Artemi mylse ctivity ws more thn 50% of the totl mylse ctivity we mesured in the metmorphic lrve. A high contribution Ž up to 23%. of mylse ctivity by prey orgnisms lso hs been reported in nother coldwter crnivorous fish, the wlleye pollock Ž Oozeki nd Biley, The estimted contribution of Artemi to the totl ctivities of the other three enzymes in metmorphic lrve of Atlntic hlibut ws reltively smll Ž 2 10%., nd the contribution to trypsin nd

9 ( ) A. Gwlick et l.raquculture lipse levels we recorded re similr to those found in wlleye pollock lrve fed rotifers Ž Oozeki nd Biley, nd in striped bss lrve fed Artemi nuplii ŽOzkizilcik et l., Enzymes other thn mylse in Artemi my be of indirect importnce in the young fish s digestive processes s hs been proposed for sebss lrve ŽMunill-Morn et l., 1990; Kolkovski et l., 1997b.. For instnce, the smll contribution of exogenous trypsin my be sufficient to llow utolytic proteolysis of Artemi in the fish s digestive system Ž Semin et l., 1980., which, in turn, my ctivte enzyme zymogens ŽMunill- Morn et l., 1990; Oozeki nd Biley, or digestive hormones ŽKolkovski et l., in the fish. The proposed commercil dvntge of feeding yolk-sc lrve s erly s dd Ž Pittmn, seems biologiclly unrelistic nd economiclly disdvntgeous Ž Hrboe nd Mngor-Jensen, becuse of low digestive cpcities of Atlntic hlibut to process exogenous nutrients before 230 dd. The ontogenetic sequence of digestive system development ppers to be geneticlly progrmmed in fishes ŽDbrow- ski, 1986; Buddington nd Dimond, 1989.; thus, only limited diet-relted mnipultion of digestive bilities my be possible ŽBuddington et l., 1987; Collie nd Ferrris, 1995; Peres et l., Becuse Atlntic hlibut lrve require exogenous nutrients only from 200 dd Ž Finn et l., 1995., erlier introduction of live food orgnisms increses the risk of punctured yolk-scs Ž Finn et l., nd lters the microflor of the intestine Ž Bergh et l., Conclusion The results of this study indicte tht yolk-sc lrve of Atlntic hlibut possess the bility to digest nd bsorb exogenous nutrients by 230 dd. The observed pttern of enzyme ctivities suggests tht first feeding should be initited fter 230 dd but not lter thn 276 dd to void the thret of strvtion. Although the lrve seem to be histomorphologiclly Ž Kjørsvik nd Reiersen, nd behviorlly ŽSkiftesvik et l., prepred for feeding by 180 dd, our enzymtic dt indicte tht the lrve re not redy for first feeding until 230 dd. Overll, our findings on the ptterns of digestive enzyme ctivity in dd hlibut lrve re consistent with recent feeding experiments Ž Hrboe nd Mngor-Jensen, nd commercil prctice ŽShields et l., showing tht hlibut lrve feed more successfully nd grow fster when first feeding occurs fter rther thn before 230 dd. Acknowledgements We re grteful to the technicl stff t the Austevoll Aquculture Reserch Sttion for ssistnce, the Austevoll Mrin Yngelproduksjon for dontion of hlibut lrve nd two nonymous reviewers for vluble comments on previous version of the mnuscript. Finncil support ws received from the Ntionl Reserch Council Cnd, the Nturl Sciences nd Engineering Reserch Council of Cnd nd the Norwegin Reserch Council.

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