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www.elsevier.nlrlocateraqua-online

Activity of digestive enzymes in yolk-sac larvae of

ž

/

Atlantic halibut Hippoglossus hippoglossus :

indication of readiness for first feeding

Anna Gawlicka

a,)

_{, Brigitte Parent}

_{, Michael H. Horn}

_,

Neil Ross

_{, Ingegjerd Opstad}

_{, Ole J. Torrissen}

c a

Institute for Marine Biosciences, National Research Council Canada, 1411 Oxford Street, Halifax, NoÕa

Scotia, Canada B3H 3Z1

Department of Biological Science, California State UniÕersity, Fullerton, CA 92834-6850, USA

Institute of Marine Research, AusteÕoll Aquaculture Research Station, N-5392 Storebø, Norway

Received 24 March 1999; received in revised form 23 September 1999; accepted 24 September 1999

Abstract

The problem of determining when larvae should be offered food is particularly difficult in a

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species such as Atlantic halibut that has a long yolk-sac period 280–320 degree days, dd . In order to help determine at what age Atlantic halibut larvae are able to digest food, we compared the activities of key digestive enzymes in four yolk-sac stages at an age interval that has been recommended for initiation of feeding, i.e., 161–276 dd. We tested the hypothesis that digestive enzyme activities reach highest levels near the end of this age interval. Activities of trypsin, amylase, lipase and alkaline phosphatase were determined spectrophotometrically in whole yolk-sac larvae at 161, 179, 230, and 276 dd. The activities of the same enzymes were measured

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in metamorphic larvae 660 dd and in their Artemia prey to provide reference levels from a fully developed digestive system and to estimate the importance of exogenous enzymes for Atlantic

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halibut larvae. Our results showed significant P-0.001 differences in activities of all four digestive enzymes among the yolk-sac stages with a general pattern of increase from 161 to 276 dd. Trypsin activities reached their highest values at 230 dd, whereas those of lipase and alkaline phosphatase peaked at 276 dd. Amylase activities were detected only in the 230 and 276 dd stages, at statistically indistinguishable levels. Based on percentage comparisons, specific activi-ties of trypsin and amylase in whole 276-dd larvae were only 12% and 2%, respectively, of those measured in the digestive system of metamorphic larvae, whereas specific activities of lipase and

)_{Corresponding author. Department of Biological Science, California State University at Fullerton,} Fullerton, CA 92834-6850, USA. Tel.:q1-714-278-3707; fax:q1-714-278-3426; e-mail:

[email protected]

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alkaline phosphatase in 276-dd larvae were more than 50% of those determined for metamorphic larvae. The calculated contribution of enzyme activities derived from Artemia prey to the relatively high levels of enzyme activity in the digestive system of metamorphic larvae was less than 10% for all enzymes except amylase, for which the contribution was estimated to be more than 50%. The results of this study support our hypothesis that the highest digestive enzyme activities in yolk-sac larvae are reached by 230–276 dd, i.e., near the end of the age interval recommended for first feeding. The observed pattern of enzyme activities suggests that feeding of Atlantic halibut larvae should be initiated after 230 dd, but not later than 276 dd to avoid the threat of starvation.q_{2000 Elsevier Science B.V. All rights reserved.}

Keywords: Alkaline phosphatase; Amylase; Artemia; Lipase; Trypsin

1. Introduction

The time period during which marine fish larvae in nature begin to feed is a critical

phase in their lives because it affects their survival, growth and development see

Sanderson and Kupferberg, 1999 . Larvae switch to an exogenous food supply whenever they are developed enough to ingest and digest food and absorb the nutrients. In the

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commercial rearing of marine fish larvae, this transition from endogenous yolk to exogenous feeding is often a period of high mortality. One of the main challenges of rearing larval fishes in captivity is to find ways to minimize this mortality. Meeting this challenge requires knowledge about when the larvae of the species in question are capable of exogenous feeding and thus when they should be offered food. In turn, knowing when to initiate feeding must be based on a sound understanding of the structural and functional development of the digestive system. In other words, the larvae must be capable of digesting the food consumed if they are to survive and grow.

The challenge of rearing marine fish larvae is compounded because species vary in the time of first exogenous feeding as they exhaust their yolk supply and begin to rely increasingly on external food sources. The larvae of some common species, such as

Atlantic cod, European seabass, plaice, turbot and winter flounder Pleuronectes

. Ž .

americanus , hatch from small eggs 1–2 mm with a limited amount of yolk and have a

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short 2–6 days yolk-sac period Jobling, 1995; Rønnestad et al., 1998 . The current rearing practice for these species is to begin feeding the larvae when the mouth has

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opened but before the yolk-sac is completely resorbed Watanabe and Kiron, 1994 . In

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other species, such as Atlantic halibut, the larvae hatch from larger eggs 3–3.5 mm with a greater yolk reserve and experience a longer, temperature-dependent yolk-sac

Ž . Ž .

period, which lasts for 280 Olsen et al., 1999 to 320 Lein and Holmefjord, 1992

Ž .

degree days ddswater temperature,8C,=age, days post-hatch . Clearly, the problem of deciding when to offer food to larvae with such an extended yolk-sac period is different and arguably more difficult than for larvae with a much shorter yolk-sac period. This problem is particularly acute in Atlantic halibut for which almost nothing is known about the size at first feeding in the wild.

Several approaches have been used to determine when feeding should be initiated in Atlantic halibut larvae, but have resulted in discrepancies for the recommended age for

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. Ž .

Reiersen, 1992 , morphology and behavior Blaxter et al., 1983; Pittman et al., 1990b ,

Ž . Ž . Ž

algal uptake Reitan et al., 1994 , relative protein synthesis RNArDNA ratio Pittman

et al., 1990a; Skiftesvik et al., 1991 and on respiration, nitrogen and energy metabolism

Ž_{Finn et al., 1995 in developing yolk-sac larvae recommend that feeding should be}.

started at age 150–180 dd, i.e., when about 50–30% of the yolk-sac remains. Feeding at

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this early age would be advantageous in a commercial sense Pittman, 1991 because the added nutrient intake would accelerate growth and shorten the time of production. The results, however, of feeding experiments show that Atlantic halibut larvae should be

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offered food at a later age interval, either 200–265 dd Lein and Holmefjord, 1992 ,

Ž . Ž .

215–240 dd Reitan et al., 1994 or 260–290 dd Harboe and Mangor-Jensen, 1998 .

Although the current practice is to begin feeding larvae around 220–230 dd Gara et al.,

1998 , the initial food uptake remains low and represents the main bottleneck in

Ž .

intensive rearing Shields et al., 1999 . Recent research has demonstrated that initial

feeding success can be increased by postponing first feeding to 260–290 dd Harboe and

Mangor-Jensen, 1998 . Although the feeding at this later age may be advantageous in an

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economic sense Harboe and Mangor-Jensen, 1998 , earlier work by Hjelmeland et al.

Ž1996 recommended that, based on trypsinogen. rtrypsin content, feeding should be initiated no later than 280 dd in order to avoid starvation. Solving the dilemma of time of first feeding in Atlantic halibut requires research on the poorly known ontogenetic sequence of digestive enzyme activity in order to determine at what age the larvae are able to digest and absorb exogenous nutrients.

The present study was designed to compare the activities of key digestive enzymes in four yolk-sac stages of Atlantic halibut larvae representing largely the age interval that has been recommended for initiation of feeding, i.e., 161–276 dd. We tested the hypothesis that digestive enzyme activities reach highest levels near the end of this age interval. If digestive enzyme activity is a good indicator of larval digestive capacity, then the time of highest activity should indicate when the larvae have become physio-logically ready to process exogenous food. We measured the levels of digestive enzyme

Ž .

activity in metamorphic larvae 660 dd to provide reference levels from a more highly developed digestive system while recognizing that metamorphic larvae may exhibit higher levels of digestive enzyme activity than the yolk-sac larvae in part because enzymes of their prey may contribute to total enzyme activity. In order to estimate the importance of exogenous enzymes for Atlantic halibut larvae, we calculated the relative contribution of enzyme activities derived from Artemia prey to those measured in the digestive system of metamorphic larvae.

2. Materials and methods

2.1. Rearing of larÕae

Yolk-sac larvae of Atlantic halibut, Hippoglossus hippoglossus, were obtained and reared following the standard protocol used at the Austevoll Aquaculture Research

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Eggs were stripped from one female and fertilized with milt from one male. After fertilization, eggs were incubated at 68C in 250-l conical tanks with a slow upwelling inflow. One day before hatching, eggs were moved to one 5-m3 upwelling silo supplied

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with sand-filtered seawater 33.4–34.7‰ at 6–78C and kept in darkness. Water flow was 2 lrmin for the first 2 weeks and 4 lrmin thereafter. Larvae hatched after 84 dd. By 161 dd, survival was 87% and remained high during the second half of the yolk-sac period, but only 5% of larvae survived by 276 dd because of a breakdown of the main water pump. An additional batch of yolk-sac larvae was obtained from a commercial

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flatfish hatchery Austevoll Marin Yngelprodusjon , and reared in circular fiberglass

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tanks Harboe et al., 1998 supplied with ‘‘green seawater’’ Næss et al., 1995 at 128C and an inflow of 1 lrmin. Feeding was initiated on 265 dd with enriched brine shrimp

ŽArtemia nauplii McEvoy et al., 1998 distributed three times a day at a rate of 1000. Ž .

naupliirlrday. Larvae were also given natural zooplankton between 6 and 20 days of

Ž .

feeding as recommended by Næss et al. 1995 . From 650 dd, the feeding rate and water inflow were increased progressively to 6000 naupliirlrday and 6 lrmin, respectively. 2.2. Sampling of larÕae

Yolk-sac larvae were collected at 161, 179, 230 and 276 dd i.e., 26, 29, 37 and 44

days post-hatch, dph and euthanized individually with an overdose of metomidate

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hydrochloride 0.01 grl, Wildlife Pharmaceuticals, Fort Collins, CO, USA . Larvae were placed on a glass surface kept on ice and examined under a dissecting microscope to eliminate individuals with abnormally formed jaws. One hundred normally formed larvae were grouped by tens, dried of residual water with paper toweling, and then

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frozen in liquid nitrogen. At 161, 179, 230 and 276 dd, mean wet vs. dry, 48 h at 608C

Ž . Ž . Ž . Ž .

body masses of individual, unfrozen larvae ns10 were 6.4 0.5 , 6.6 0.6 , 5.8 0.8

Ž . Ž .

and 5.3 0.5 mg, respectively, with SE -10% in each case.

Ten metamorphic larvae with normal pigmentation, asymmetric bodies and

com-Ž .

pletely migrated left eye were sampled at 660 dd 78 dph after 34 days of feeding.

Larvae were dried with paper toweling, weighed individually mean wet body mass of

56.0"9.0 mg and dissected on a glass surface kept on ice and 10 whole digestive

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systems including liver and pancreas containing Artemia were frozen. Photographs of five additional metamorphic larvae from the same group were taken in order to provide an estimate of the number of Artemia nauplii in a digestive system. Samples of live Artemia nauplii were collected on a 80-mm mesh sieve and transferred to five replicate cryo-vials with ca. 14,000 Artemia nauplii per vial before freezing in liquid nitrogen. All samples were stored aty808C until processed.

2.3. Enzyme assays

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Frozen whole yolk-sac larvae ns5 pooled samples of 10 larvae and digestive

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systems of metamorphic larvae ns5 were partially thawed, weighed and

homoge-Ž .

nized on ice in five volumes of 0.2 M NaCl wrv using a motorized teflon pestle. Frozen samples of Artemia were homogenized on ice in two volumes of 0.2 M NaCl. This saline solution was chosen because it is naturally present in the lumen of the

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digestive system of marine fishes. The suspensions were centrifuged at 12,000=g for 5 min and the supernatants placed on ice and used immediately for spectrophotometric determination of enzyme activities and soluble protein content following the protocols optimized on adult guts and described below. All assays were carried out in triplicate at

Ž . Ž

room temperature 238C using a Thermomax microplate spectrophotometer Molecular

Devices, Sunnyvale, CA, USA . Blanks were used to account for non-enzymatic hydrolysis of substrates. The concentrations given correspond to those in the final incubation mediums.

Ž . Ž .

Trypsin E.C. 3.4.21.4 activity was assayed following Erlanger et al. 1961 .

Homogenates were incubated with 1 mM BAPNA N-a-benzoyl-L-arginine p-nitro-.

anilide hydrochloride, Boehringer Mannheim, cat. no. 775 819 in 25 mM ammonium bicarbonate buffer, pH 7.8, and the increase in absorbance was measured at 405 nm for 30 min.

Ž .

Amylase E.C. 3.2.1.1 was measured according to the Somogyi–Nelson procedure

ŽSomogyi, 1952 . Starch substrate was prepared by boiling 1% soluble starch Sigma,. Ž .

S2630 in 0.8 M sodium citrate buffer, pH 7.0, for 5 min. Samples were incubated with 0.5% starch in 0.2 M sodium citrate at pH 7.0, a pH considered optimal for amylase in

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adult Atlantic halibut Glass et al., 1987 . The incubation was stopped after 6 h by adding 0.2 volumes of 1 M NaOH and two volumes of the first Somogyi–Nelson reagent. Reducing sugars were determined by measuring the changes in absorbance at 650 nm.

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Lipase nonspecific, E.C. 3.1.1.- activity was measured according to a modified

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method of Albro et al. 1985 . Homogenates were incubated with 0.4 mM p-nitrophenyl

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myristate Sigma, N2502 in 24 mM ammonium bicarbonate, pH 7.8, containing 0.5% Triton X-100 as an emulsifying agent. The change in absorbance was measured at 405 nm for 30 min.

Ž . Ž .

Alkaline phosphatase E.C. 3.1.3.1 was assayed following Walter and Schutt 1974 .

¨

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Homogenates were incubated with 4 mM p-nitrophenyl phosphate Sigma, N6750 in 55 mM ammonium bicarbonate buffer with 0.6 mM MgCl , pH 7.8. The increase in2

absorbance was measured continuously for 30 min at 405 nm.

Soluble protein content in homogenates of whole larvae and digestive systems was

Ž . Ž

determined by the method of Bradford 1976 , using bovine gamma-globulin BioRad,

Mississauga, Ontario, Canada as a standard.

Concentrations of the assay products were determined experimentally based on a standard curve of absorbance vs. product concentration using the same volumes as in the assays. All activities determined for the digestive enzymes were within the dynamic range of the respective assays. Enzyme activity was expressed as specific activity

ŽmUrmg protein or tissue activity mU. Ž rmg tissue and represented nanomoles of.

product liberated during 1 min of hydrolysis per milligram of protein or milligram of

Ž . Ž .

whole larvae 161–276 dd or digestive systems 660 dd . 2.4. Statistical analyses

Mean values of enzyme activities and soluble protein contents were compared among

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that were significantly different were analyzed further by a Fisher multiple-comparison test. An a level of 0.05 was established a priori for all statistical tests.

3. Results

Significant differences in specific and tissue activities of all four digestive enzymes were found among the yolk-sac stages with a general pattern of increase from 161 to

Ž .

276 dd Table 1 . Trypsin activities reached their highest values at 230 dd, whereas those of lipase and alkaline phosphatase peaked at 276 dd. Amylase activities were detected only in the 230 and 276 dd stages, at statistically indistinguishable levels. When expressed in mUrmg protein, the activities of lipase and alkaline phosphatase decreased significantly from the 161- to the 179-dd stage, paralleling an increase in soluble protein content. Based on percentage comparisons, specific activities of trypsin and amylase in whole 276-dd larvae were only 12% and 2%, respectively, of those measured in the digestive systems of metamorphic larvae, whereas specific activities of lipase and alkaline phosphatase in 276-dd larvae were more than 50% of those determined for metamorphic larvae.

Mean specific activities of the digestive enzymes in Artemia nauplii are given in

Ž .

Table 2. At least 200 nauplii range 200–300 were found in the digestive system of

Table 1

Activities of digestive enzymes and content of soluble proteins in whole larvae at four yolk-sac stages

Ž . Ž . U

161–276 dd and in the whole digestive system of metamorphic larvae 660 dd of Atlantic halibut

Ž .

EnzymerProtein Larvae dd

Yolk-sac Metamorphic

161 179 230 276 660

( )

Specific actiÕities mUrmg protein

a b d c

Trypsin 3.4"0.1 9.3"0.8 16.7"0.7 11.6"0.5 98.6"18.6

a a b b

Amylase 0.0"0.0 0.0"0.0 27.0"6.2 32.9"6.1 1633.6"401.0

c a b d

Lipase 15.7"0.9 9.5"0.4 13.4"0.5 22.6"0.7 37.3"2.8

b a b c

Alkaline phosphatase 34.2"2.1 21.9"1.1 38.5"1.1 61.2"3.4 111.9"7.2

( )

Tissue actiÕities mUrmg tissue

a c d b

Trypsin 0.1"0.0 0.3"0.0 0.4"0.0 0.2"0.0 4.4"1.8

a a b b

Amylase 0.0"0.0 0.0"0.0 0.5"0.1 0.6"0.1 74.1"20.2

a a a b

Lipase 0.3"0.0 0.3"0.0 0.3"0.0 0.4"0.0 1.7"0.2

a a b c

Alkaline phosphatase 0.6"0.1 0.6"0.1 0.8"0.0 1.0"0.0 5.0"0.5

( )

Soluble protein contents mgrmg tissue

a c b a

16.8"1.2 27.1"1.1 21.2"1.0 17.2"1.0 44.9"2.8 U

Means"1 SE ns5 pooled samples of 10 yolk-sac larvae or ns5 digestive systems of metamorphic

. Ž

larvae were compared among yolk-sac stages only. Specific activities, Fs85.3, trypsin; 20.5, amylase; 75.8, lipase; 58.2, alkaline phosphatase. Tissue activities, Fs82.2, trypsin; 11.4, amylase; 32.7, lipase; 29.0, alkaline phosphatase. Soluble protein contents, Fs24.1. P-0.001 and dfs19 in all cases, except, dfs17

for amylase . Location and number of significantly different means are designated by different superscript

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Table 2

Ž .

Specific activities mUrmg protein of digestive enzymes measured in Artemia prey and their percentage contribution to the total specific activity of these enzymes calculated for the whole digestive system of metamorphic larvae of Atlantic halibut containing 200 Artemia nauplii per larva.a _{See Section 2.2 for} additional details

Enzyme Activity in Calculated activity % Contribution of

Artemia _{200 Artemia} _{Digestive system} Artemia enzymes

Trypsin 52.6"6.7 4.2 50.3 8.4

Amylase 5,449"30 435.9 833.1 52.3

Lipase 6.3"0.7 0.5 19.0 2.6

Alkaline phosphatase 68.8"9.2 5.5 57.1 9.6

a _Ž

Means"1 SE ns3 pooled samples of ca. 14,000 Artemia nauplii or ns3 digestive systems of

metamorphic larvae .

metamorphic larvae. The percentage contribution of 200 such nauplii to total enzymatic activity in the digestive system of these larvae was less than 10% for all enzymes except amylase for which the contribution was estimated to be more than 50%.

4. Discussion

The results of this study support our hypothesis that the highest digestive enzyme

Ž .

activities are reached near the end of the age interval 161–276 dd recommended for first feeding in Atlantic halibut larvae. We found, however, differences in the time and amount of change in activity among the four enzymes studied, and these may reflect the extended sequence of functional development in the digestive system of a fish, such as Atlantic halibut, with a long yolk-sac period.

The pattern of trypsin activity showing a peak at the 230-dd yolk-sac stage followed by a decrease at 276 dd suggests that larvae of Atlantic halibut should be fed in this interval of development. The peak in trypsin activity indicates increased functionality of

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the pancreas Segner et al., 1994; Kurokawa and Suzuki, 1996 , whereas the subsequent

decline may be a sign of pancreatic tissue degeneration from starvation Kjørsvik et al.,

1991; Ueberschar, 1993; Tanaka et al., 1996 . This pattern appears to be consistent with

¨

increased trypsinogenrtrypsin content at 245–265 dd and its decrease by 280 dd

Ž .

reported for Atlantic halibut Hjelmeland et al., 1996 . In other marine fish larvae, all with short yolk-sac periods, first feeding correlates with increased trypsinogenrtrypsin

Ž .

content Hjelmeland et al., 1984; Pedersen et al., 1987; Kurokawa and Suzuki, 1996

and increased trypsin activity Lauff and Hofer, 1984; Ueberschar, 1993; Zambonino

¨

Infante and Cahu, 1994; Oozeki and Bailey, 1995 . For Atlantic halibut larvae, the pattern appears to be similar, but the timeframe is necessarily extended because of the long yolk resorption period.

The pattern of amylase activity, showing no detectable levels at 161 and 179 dd and relatively low levels at 230 and 276 dd compared to other values reported for marine

fish larvae at first feeding Zambonino Infante and Cahu, 1994, 1999; Oozeki and

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Ž .

carbohydrates. This limited ability may explain the low efficiency 1–5% with which

Atlantic halibut assimilate microalgae throughout the yolk-sac period Reitan et al.,

1994 . The inefficient use of carbohydrates by Atlantic halibut and other carnivorous marine fishes that inhabit cold waters is not surprising given that amylase activity is

Ž .

known Munilla-Moran and Saborido-Rey, 1996 to be low at 5

´

8C, which is one of the

Ž .

standard rearing temperatures 5–68C of halibut yolk-sac larvae. Larvae of marine fish,

Ž .

including Atlantic halibut see below , that have been eating zooplankton, however, may

Ž .

exhibit higher amylase activity. In walleye pollock Theragra chalcogramma , Oozeki

Ž .

and Bailey 1995 showed that most of this increased activity originates from the zooplankton prey, whose amylases exhibit lower temperature optima than those of fish

ŽMayzaud, 1985 ..

The pattern of continuous increase in lipase activity through the yolk-sac period in Atlantic halibut suggests that the larvae are prepared to feed on lipid-rich zooplankton by 276 dd. This high lipase activity in the later stages of yolk-sac resorption coincides

with the period of increased use of lipids for energy by Atlantic halibut larvae Rainuzzo

et al., 1992; Finn et al., 1995; Rønnestad et al., 1995 and agrees with recent studies on

lipases in other marine fish larvae Ozkizilcik et al., 1996; Zambonino Infante and Cahu,

1999 . Further research is required to verify that lipolytic capacities in Atlantic halibut

Ž .

larvae are limited by low bile salt production as suggested by Rønnestad et al. 1995 . The pattern of continuous increase in activity of alkaline phosphatase through the yolk-sac period in Atlantic halibut suggests that high absorptive capacities of the intestine are attained by 276 dd. Alkaline phosphatase activity has been associated with

Ž .

absorption of extracellular nutrients see Zueva et al., 1993 and its increase used as an indicator of the onset of absorptive function in the intestinal epithelium of fish larvae

ŽCousin et al., 1987; Zambonino Infante and Cahu, 1994; Gawlicka et al., 1995; Baglole

et al., 1998 . A decrease in alkaline phosphatase activity usually accompanies starvation

ŽCousin et al., 1987 or the feeding of an inadequate diet Cahu and Zambonino Infante,. Ž .

1994; Gawlicka et al., 1996 . An increase in activity of this enzyme, however, also has

Ž .

been observed in malnourished seabass larvae Zambonino Infante and Cahu, 1994 . Whether the high levels of alkaline phosphatase in the unfed halibut larvae in our study signaled higher absorptive capacities or starvation remains to be determined by histolog-ical examination of the intestine in 276 dd larvae.

Our results suggest that amylase but not trypsin, lipase or alkaline phosphatase from Artemia prey contributes importantly to digestive capacity in metamorphic larvae of Atlantic halibut. The high amylase activity we recorded in these 660 dd larvae parallels

the activity level of this enzyme in metamorphic seabass larvae fed Artemia Zambonino

Infante and Cahu, 1994 . Artemia nauplii are herbivores and are expected to have high amylase levels in order to digest the carbohydrates found in the microalgae they are fed

Ž .

in culture Semain et al., 1980 . Thus, not surprisingly, the minimum calculated contribution of Artemia amylase activity was more than 50% of the total amylase

Ž .

activity we measured in the metamorphic larvae. A high contribution up to 23% of amylase activity by prey organisms also has been reported in another coldwater

Ž .

carnivorous fish, the walleye pollock Oozeki and Bailey, 1995 . The estimated contribu-tion of Artemia to the total activities of the other three enzymes in metamorphic larvae

Ž .

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lipase levels we recorded are similar to those found in walleye pollock larvae fed rotifers

ŽOozeki and Bailey, 1995 and in striped bass larvae fed Artemia nauplii Ozkizilcik et. Ž .

al., 1996 . Enzymes other than amylase in Artemia may be of indirect importance in the

young fish’s digestive processes as has been proposed for seabass larvae Munilla-Moran

´

et al., 1990; Kolkovski et al., 1997b . For instance, the small contribution of exogenous trypsin may be sufficient to allow autolytic proteolysis of Artemia in the fish’s digestive

Ž . Ž

system Semain et al., 1980 , which, in turn, may activate enzyme zymogens

Munilla-. Ž

Moran et al., 1990; Oozeki and Bailey, 1995 or digestive hormones Kolkovski et al.,

´

1997a in the fish.

The proposed commercial advantage of feeding yolk-sac larvae as early as 150–180

Ž .

dd Pittman, 1991 seems biologically unrealistic and economically disadvantageous

ŽHarboe and Mangor-Jensen, 1998 because of low digestive capacities of Atlantic.

halibut to process exogenous nutrients before 230 dd. The ontogenetic sequence of

digestive system development appears to be genetically programmed in fishes

Dabrow-.

ski, 1986; Buddington and Diamond, 1989 ; thus, only limited diet-related manipulation

of digestive abilities may be possible Buddington et al., 1987; Collie and Ferraris, 1995;

Peres et al., 1998 . Because Atlantic halibut larvae require exogenous nutrients only

´

Ž .

from 200 dd Finn et al., 1995 , earlier introduction of live food organisms increases the

Ž .

risk of punctured yolk-sacs Finn et al., 1995 and alters the microflora of the intestine

Ž_{Bergh et al., 1994 .}.

5. Conclusion

The results of this study indicate that yolk-sac larvae of Atlantic halibut possess the ability to digest and absorb exogenous nutrients by 230 dd. The observed pattern of enzyme activities suggests that first feeding should be initiated after 230 dd but not later than 276 dd to avoid the threat of starvation. Although the larvae seem to be

Ž . Ž

histomorphologically Kjørsvik and Reiersen, 1992 and behaviorally Skiftesvik et al.,

1994 prepared for feeding by 180 dd, our enzymatic data indicate that the larvae are not ready for first feeding until 230 dd. Overall, our findings on the patterns of digestive enzyme activity in 161–276 dd halibut larvae are consistent with recent feeding

Ž . Ž

experiments Harboe and Mangor-Jensen, 1998 and commercial practice Shields et al.,

1999 showing that halibut larvae feed more successfully and grow faster when first feeding occurs after rather than before 230 dd.

Acknowledgements

We are grateful to the technical staff at the Austevoll Aquaculture Research Station for assistance, the Austevoll Marin Yngelproduksjon for donation of halibut larvae and two anonymous reviewers for valuable comments on a previous version of the manuscript. Financial support was received from the National Research Council Canada, the Natural Sciences and Engineering Research Council of Canada and the Norwegian Research Council.

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Erlanger, B.F., Kokowsky, N., Cohen, W., 1961. The preparation and properties of two new chromogenic substrates of trypsin. Arch. Biochem. Biophys. 95, 271–278.

Finn, R.N., Rønnestad, I., Fyhn, H.J., 1995. Respiration, nitrogen and energy metabolism of developing

Ž .

yolk-sac larvae of Atlantic halibut Hippoglossus hippoglossus L. . Comp. Biochem. Physiol. 111A,

647–671.

Gara, B., Shields, R.J., McEvoy, L., 1998. Feeding strategies to achieve correct metamorphosis of Atlantic halibut, Hippoglossus hippoglossus L., using enriched Artemia. Aquacult. Res. 29, 935–948.

Gawlicka, A., Teh, S.J., Hung, S.S.O., Hinton, D.E., de la Noue, J., 1995. Histological and histochemical¨ changes in the digestive tract of white sturgeon larvae during ontogeny. Fish Physiol. Biochem. 14, 357–371.

Gawlicka, A., McLaughlin, L., Hung, S.S.O., de la Noue, J., 1996. Limitations of carrageenan microbound¨ diets for feeding white sturgeon, Acipenser transmontanus, larvae. Aquaculture 141, 245–265.

Glass, H.J., MacDonald, L., Stark, J.R., 1987. Metabolism in marine flatfish: IV. Carbohydrate and protein

Ž .

digestion in Atlantic halibut Hippoglossus hippoglossus L. . Comp. Biochem. Physiol. 86B, 281–289. Harboe, T., Mangor-Jensen, A., 1998. Time of first feeding of Atlantic halibut, Hippoglossus hippoglossus L.,

larvae. Aquacult. Res. 29, 913–918.

Harboe, T., Huse, S., Øie, G., 1994a. Effect of egg disinfection on yolk-sac and first feeding stages of halibut

ŽHippoglossus hippoglossus L. larvae. Aquaculture 119, 157–165..

Harboe, T., Tuene, S., Mangor-Jensen, A., Rabbe, H., Huse, I., 1994b. Design and operation of an incubator for yolk-sac larvae of Atlantic halibut. Prog. Fish-Cult. 56, 188–193.

Harboe, T., Mangor-Jensen, A., Naas, K.E., Næss, T., 1998. A tank design for first feeding of Atlantic halibut,

Hippoglossus hippoglossus L., larvae. Aquacult. Res. 29, 919–923.

Hjelmeland, K., Huse, I., Jørgensen, T., Molvik, G., Raa, J., 1984. Trypsin and trypsinogen as indices of

Ž .

growth and survival potential of cod Gadus morhua L. larvae. In: Dahl, E., Danielssen, D.S., Moksness,

Ž .

E., Solemdal, P. Eds. , The Propagation of Cod Gadus morhua L. University of Tromsø, Tromsø, Norway, pp. 189–202.

(11)

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Ž .

Jobling, M., 1995. Development of eggs and larvae. In: Jobling, M. Ed. , Environmental Biology of Fishes, Chapter 10. Chapman & Hall, London England, pp. 357–390.

Kjørsvik, E., Reiersen, A.L., 1992. Histomorphology of the early yolk-sac larvae of the Atlantic halibut

ŽHippoglossus hippoglossus L. — an indication of the timing of functionality. J. Fish Biol. 41, 1–19..

Kjørsvik, E., Van Der Meeren, T., Kryvi, H., Arnfinnson, J., Kvenseth, P.G., 1991. Early development of the digestive tract of cod larvae, Gadus morhua L., during start-feeding and starvation. J. Fish Biol. 28, 1–15. Kolkovski, S., Koven, W., Tandler, A., 1997a. The mode of action of Artemia in enhancing utilization of

microdiet by gilthead seabream Sparus aurata larvae. Aquaculture 155, 193–205.

Kolkovski, S., Tandler, A., Izquierdo, M.S., 1997b. Effects of live food and dietary digestive enzymes on the

Ž .

efficiency of microdiets for seabass Dicentrarchus labrax larvae. Aquaculture 148, 313–322.

Lauff, M., Hofer, R., 1984. Proteolytic enzymes in fish development and the importance of dietary enzymes. Aquaculture 37, 335–346.

Lein, I., Holmefjord, I., 1992. Age at first feeding of Atlantic halibut larvae. Aquaculture 105, 157–164. Mayzaud, O., 1985. Purification and kinetic properties of thea-amylase from the copepod Acartia clausi.

Comp. Biochem. Physiol. 82B, 725–730.

McEvoy, L.A., Næss, T., Bell, J.G., Lie, Ø., 1998. Lipid and fatty acid composition of normal and

Ž .

malpigmented Atlantic halibut Hippoglossus hippoglossus fed enriched Artemia: a comparison with fry fed wild copepods. Aquaculture 163, 237–250.

Munilla-Moran, R., Saborido-Rey, F., 1996. Digestive enzymes in marine species: II. Amylase activities in gut´

Ž . Ž . Ž .

from seabream Sparus aurata , turbot Scophthalmus maximus and redfish Sebastes mentella . Comp. Biochem. Physiol. 113B, 827–834.

Munilla-Moran, R., Stark, J.R., Barbour, A., 1990. The role of exogenous enzymes in digestion in cultured´

Ž .

turbot larvae Scophthalmus maximus L. . Aquaculture 88, 337–350.

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Næss, T., Germain-Henry, M., Naas, K.E., 1995. First feeding of Atlantic halibut Hippoglossus hippoglossus using different combination of Artemia and wild zooplankton. Aquaculture 130, 235–250.

Olsen, Y., Evjemo, J.O., Olsen, A., 1999. Status of the cultivation technology for production of Atlantic

Ž .

halibut Hippoglossus hippoglossus juveniles in NorwayrEurope. Aquaculture 176, 3–13.

Oozeki, Y., Bailey, K.M., 1995. Ontogenetic development of digestive enzyme activities in larval walleye pollock, Theragra chalcogramma. Mar. Biol. 122, 177–186.

Ozkizilcik, S., Chu, F.-L.E., Place, A.R., 1996. Ontogenetic changes of lipolytic enzymes in striped bass

ŽMorone saxatilis . Comp. Biochem. Physiol. 113B, 631–637..

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Ž .

larval herring Clupea harengus digesting copepod nauplii. Mar. Biol. 94, 171–181.

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trypsin and amylase in seabass Dicentrarchus labrax larvae. Fish Physiol. Biochem. 19, 145–152.

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Pittman, K.A., 1991. Aspects of the early life history of the Atlantic halibut Hippoglossus hippoglossus L. : embryonic and larval development and the effects of temperature. PhD thesis. University of Bergen, Bergen, Norway.

Pittman, K., Bergh, Ø., Opstad, I., Skiftesvik, A.B., Skjolddal, L., Strand, H., 1990a. Development of eggs and

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yolk-sac larvae of halibut Hippoglossus hippoglossus L. . J. Appl. Ichthyol. 6, 142–160.

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Hippoglossus hippoglossus L. larvae. J. Fish Biol. 37, 455–472.

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Atlantic halibut Hippoglossus hippoglossus . J. Fish Biol. 44, 303–310.

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developing eggs and larvae of European seabass Dicentrarchus labrax . Aquaculture 161, 157–170. Sanderson, S.L., Kupferberg, S.J., 1999. Development and evolution of aquatic larval feeding mechanisms. In:

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Table 2

Ž .

Enzyme Activity in Calculated activity % Contribution of

Artemia _{200 Artemia} _{Digestive system} Artemia enzymes

Trypsin 52.6"6.7 4.2 50.3 8.4

Amylase 5,449"30 435.9 833.1 52.3

Lipase 6.3"0.7 0.5 19.0 2.6

Alkaline phosphatase 68.8"9.2 5.5 57.1 9.6

a _Ž

Means"1 SE ns3 pooled samples of ca. 14,000 Artemia nauplii or ns3 digestive systems of

metamorphic larvae .

metamorphic larvae. The percentage contribution of 200 such nauplii to total enzymatic

activity in the digestive system of these larvae was less than 10% for all enzymes except

amylase for which the contribution was estimated to be more than 50%.

4. Discussion

The results of this study support our hypothesis that the highest digestive enzyme

Ž

.

activities are reached near the end of the age interval 161–276 dd recommended for

first feeding in Atlantic halibut larvae. We found, however, differences in the time and

amount of change in activity among the four enzymes studied, and these may reflect the

extended sequence of functional development in the digestive system of a fish, such as

Atlantic halibut, with a long yolk-sac period.

The pattern of trypsin activity showing a peak at the 230-dd yolk-sac stage followed

by a decrease at 276 dd suggests that larvae of Atlantic halibut should be fed in this

interval of development. The peak in trypsin activity indicates increased functionality of

Ž

.

the pancreas Segner et al., 1994; Kurokawa and Suzuki, 1996 , whereas the subsequent

Ž

decline may be a sign of pancreatic tissue degeneration from starvation Kjørsvik et al.,

.

1991; Ueberschar, 1993; Tanaka et al., 1996 . This pattern appears to be consistent with

¨

increased trypsinogen

r

trypsin content at 245–265 dd and its decrease by 280 dd

Ž

.

reported for Atlantic halibut Hjelmeland et al., 1996 . In other marine fish larvae, all

with short yolk-sac periods, first feeding correlates with increased trypsinogen

r

trypsin

Ž

.

content Hjelmeland et al., 1984; Pedersen et al., 1987; Kurokawa and Suzuki, 1996

Ž

and increased trypsin activity Lauff and Hofer, 1984; Ueberschar, 1993; Zambonino

¨

.

Infante and Cahu, 1994; Oozeki and Bailey, 1995 . For Atlantic halibut larvae, the

pattern appears to be similar, but the timeframe is necessarily extended because of the

long yolk resorption period.

The pattern of amylase activity, showing no detectable levels at 161 and 179 dd and

relatively low levels at 230 and 276 dd compared to other values reported for marine

Ž

fish larvae at first feeding Zambonino Infante and Cahu, 1994, 1999; Oozeki and

.

(2)

Ž

.

carbohydrates. This limited ability may explain the low efficiency 1–5% with which

Ž

Atlantic halibut assimilate microalgae throughout the yolk-sac period Reitan et al.,

.

1994 . The inefficient use of carbohydrates by Atlantic halibut and other carnivorous

marine fishes that inhabit cold waters is not surprising given that amylase activity is

Ž

.

known Munilla-Moran and Saborido-Rey, 1996 to be low at 5

´

8 C, which is one of the

Ž

.

standard rearing temperatures 5–6

8 C of halibut yolk-sac larvae. Larvae of marine fish,

Ž

.

including Atlantic halibut see below , that have been eating zooplankton, however, may

Ž

.

exhibit higher amylase activity. In walleye pollock Theragra chalcogramma , Oozeki

Ž

.

and Bailey 1995 showed that most of this increased activity originates from the

zooplankton prey, whose amylases exhibit lower temperature optima than those of fish

Ž

Mayzaud, 1985 .

.

The pattern of continuous increase in lipase activity through the yolk-sac period in

Atlantic halibut suggests that the larvae are prepared to feed on lipid-rich zooplankton

by 276 dd. This high lipase activity in the later stages of yolk-sac resorption coincides

Ž

with the period of increased use of lipids for energy by Atlantic halibut larvae Rainuzzo

.

et al., 1992; Finn et al., 1995; Rønnestad et al., 1995 and agrees with recent studies on

Ž

lipases in other marine fish larvae Ozkizilcik et al., 1996; Zambonino Infante and Cahu,

.

1999 . Further research is required to verify that lipolytic capacities in Atlantic halibut

Ž

.

larvae are limited by low bile salt production as suggested by Rønnestad et al. 1995 .

The pattern of continuous increase in activity of alkaline phosphatase through the

yolk-sac period in Atlantic halibut suggests that high absorptive capacities of the

intestine are attained by 276 dd. Alkaline phosphatase activity has been associated with

Ž

.

absorption of extracellular nutrients see Zueva et al., 1993 and its increase used as an

indicator of the onset of absorptive function in the intestinal epithelium of fish larvae

Ž

Cousin et al., 1987; Zambonino Infante and Cahu, 1994; Gawlicka et al., 1995; Baglole

.

et al., 1998 . A decrease in alkaline phosphatase activity usually accompanies starvation

Ž

Cousin et al., 1987 or the feeding of an inadequate diet Cahu and Zambonino Infante,

.

Ž

.

1994; Gawlicka et al., 1996 . An increase in activity of this enzyme, however, also has

Ž

.

been observed in malnourished seabass larvae Zambonino Infante and Cahu, 1994 .

Whether the high levels of alkaline phosphatase in the unfed halibut larvae in our study

signaled higher absorptive capacities or starvation remains to be determined by

histolog-ical examination of the intestine in 276 dd larvae.

Our results suggest that amylase but not trypsin, lipase or alkaline phosphatase from

Artemia prey contributes importantly to digestive capacity in metamorphic larvae of

Atlantic halibut. The high amylase activity we recorded in these 660 dd larvae parallels

Ž

the activity level of this enzyme in metamorphic seabass larvae fed Artemia Zambonino

.

Infante and Cahu, 1994 . Artemia nauplii are herbivores and are expected to have high

amylase levels in order to digest the carbohydrates found in the microalgae they are fed

Ž

.

in culture

Semain et al., 1980 . Thus, not surprisingly, the minimum calculated

contribution of Artemia amylase activity was more than 50% of the total amylase

Ž

.

activity we measured in the metamorphic larvae. A high contribution up to 23% of

amylase activity by prey organisms also has been reported in another coldwater

Ž

.

carnivorous fish, the walleye pollock Oozeki and Bailey, 1995 . The estimated

contribu-tion of Artemia to the total activities of the other three enzymes in metamorphic larvae

Ž

.

(3)

lipase levels we recorded are similar to those found in walleye pollock larvae fed rotifers

Ž

Oozeki and Bailey, 1995 and in striped bass larvae fed Artemia nauplii Ozkizilcik et

.

Ž

.

al., 1996 . Enzymes other than amylase in Artemia may be of indirect importance in the

Ž

young fish’s digestive processes as has been proposed for seabass larvae Munilla-Moran

´

.

et al., 1990; Kolkovski et al., 1997b . For instance, the small contribution of exogenous

trypsin may be sufficient to allow autolytic proteolysis of Artemia in the fish’s digestive

Ž

.

Ž

system Semain et al., 1980 , which, in turn, may activate enzyme zymogens

Munilla-.

Ž

Moran et al., 1990; Oozeki and Bailey, 1995 or digestive hormones Kolkovski et al.,

´

.

1997a in the fish.

The proposed commercial advantage of feeding yolk-sac larvae as early as 150–180

Ž

.

dd Pittman, 1991 seems biologically unrealistic and economically disadvantageous

Ž

Harboe and Mangor-Jensen, 1998 because of low digestive capacities of Atlantic

.

halibut to process exogenous nutrients before 230 dd. The ontogenetic sequence of

Ž

digestive system development appears to be genetically programmed in fishes

Dabrow-.

ski, 1986; Buddington and Diamond, 1989 ; thus, only limited diet-related manipulation

Ž

of digestive abilities may be possible Buddington et al., 1987; Collie and Ferraris, 1995;

.

Peres et al., 1998 . Because Atlantic halibut larvae require exogenous nutrients only

´

Ž

.

from 200 dd Finn et al., 1995 , earlier introduction of live food organisms increases the

Ž

.

risk of punctured yolk-sacs Finn et al., 1995 and alters the microflora of the intestine

Ž

_{Bergh et al., 1994 .}

.

5. Conclusion

The results of this study indicate that yolk-sac larvae of Atlantic halibut possess the

ability to digest and absorb exogenous nutrients by 230 dd. The observed pattern of

enzyme activities suggests that first feeding should be initiated after 230 dd but not later

than 276 dd to avoid the threat of starvation. Although the larvae seem to be

Ž

.

Ž

histomorphologically Kjørsvik and Reiersen, 1992 and behaviorally Skiftesvik et al.,

.

1994 prepared for feeding by 180 dd, our enzymatic data indicate that the larvae are not

ready for first feeding until 230 dd. Overall, our findings on the patterns of digestive

enzyme activity in 161–276 dd halibut larvae are consistent with recent feeding

Ž

.

Ž

experiments Harboe and Mangor-Jensen, 1998 and commercial practice Shields et al.,

.

1999 showing that halibut larvae feed more successfully and grow faster when first

feeding occurs after rather than before 230 dd.

Acknowledgements

We are grateful to the technical staff at the Austevoll Aquaculture Research Station

for assistance, the Austevoll Marin Yngelproduksjon for donation of halibut larvae and

two anonymous reviewers for valuable comments on a previous version of the manuscript.

Financial support was received from the National Research Council Canada, the Natural

Sciences and Engineering Research Council of Canada and the Norwegian Research

Council.

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Ž .

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Harboe, T., Huse, S., Øie, G., 1994a. Effect of egg disinfection on yolk-sac and first feeding stages of halibut

ŽHippoglossus hippoglossus L. larvae. Aquaculture 119, 157–165..

Harboe, T., Tuene, S., Mangor-Jensen, A., Rabbe, H., Huse, I., 1994b. Design and operation of an incubator for yolk-sac larvae of Atlantic halibut. Prog. Fish-Cult. 56, 188–193.

Harboe, T., Mangor-Jensen, A., Naas, K.E., Næss, T., 1998. A tank design for first feeding of Atlantic halibut, Hippoglossus hippoglossus L., larvae. Aquacult. Res. 29, 919–923.

Hjelmeland, K., Huse, I., Jørgensen, T., Molvik, G., Raa, J., 1984. Trypsin and trypsinogen as indices of

Ž .

growth and survival potential of cod Gadus morhua L. larvae. In: Dahl, E., Danielssen, D.S., Moksness,

Ž .

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(5)

Ž .

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Kjørsvik, E., Reiersen, A.L., 1992. Histomorphology of the early yolk-sac larvae of the Atlantic halibut

ŽHippoglossus hippoglossus L. — an indication of the timing of functionality. J. Fish Biol. 41, 1–19..

microdiet by gilthead seabream Sparus aurata larvae. Aquaculture 155, 193–205.

Kolkovski, S., Tandler, A., Izquierdo, M.S., 1997b. Effects of live food and dietary digestive enzymes on the

Ž .

efficiency of microdiets for seabass Dicentrarchus labrax larvae. Aquaculture 148, 313–322.

Kurokawa, T., Suzuki, T., 1996. Formation of the diffuse pancreas and the development of digestive enzyme synthesis in larvae of the Japanese flounder Paralichthys oliÕaceus. Aquaculture 141, 267–276. Lauff, M., Hofer, R., 1984. Proteolytic enzymes in fish development and the importance of dietary enzymes.

Aquaculture 37, 335–346.

Comp. Biochem. Physiol. 82B, 725–730.

McEvoy, L.A., Næss, T., Bell, J.G., Lie, Ø., 1998. Lipid and fatty acid composition of normal and

Ž .

malpigmented Atlantic halibut Hippoglossus hippoglossus fed enriched Artemia: a comparison with fry fed wild copepods. Aquaculture 163, 237–250.

Munilla-Moran, R., Saborido-Rey, F., 1996. Digestive enzymes in marine species: II. Amylase activities in gut´

Ž . Ž . Ž .

from seabream Sparus aurata , turbot Scophthalmus maximus and redfish Sebastes mentella . Comp. Biochem. Physiol. 113B, 827–834.

Munilla-Moran, R., Stark, J.R., Barbour, A., 1990. The role of exogenous enzymes in digestion in cultured´

Ž .

turbot larvae Scophthalmus maximus L. . Aquaculture 88, 337–350.

Ž .

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