. 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
Ž .
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 Ž
. 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 . Ž
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
Ž 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
Ž .
economic sense Harboe and Mangor-Jensen, 1998 , earlier work by Hjelmeland et al. Ž
. 1996 recommended that, based on trypsinogenrtrypsin 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 Ž
. Station of the Institute of Marine Research, Storebø, Norway Harboe et al., 1994a,b .
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-m
3
upwelling silo supplied Ž
. 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
Ž .
flatfish hatchery Austevoll Marin Yngelprodusjon , and reared in circular fiberglass Ž
. Ž
. 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 Ž
. Ž
. Artemia nauplii McEvoy et al., 1998 distributed three times a day at a rate of 1000
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
Ž 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
Ž .
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
Ž .
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 n s 10 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. Ž
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 Ž
. 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 at y808C until processed.
2.3. Enzyme assays Ž
. Frozen whole yolk-sac larvae
n s 5 pooled samples of 10 larvae and digestive Ž
. systems of metamorphic larvae n s 5 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
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 .
Ž 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
Ž .
Ž Somogyi, 1952 . Starch substrate was prepared by boiling 1 soluble starch Sigma,
. 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 Ž
. 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.
Ž .
Lipase nonspecific, E.C. 3.1.1.- activity was measured according to a modified Ž
. method of Albro et al. 1985 . Homogenates were incubated with 0.4 mM p-nitrophenyl
Ž .
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 .
¨
Ž .
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 in
2
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
Ž .
Ž .
mUrmg protein or tissue activity mUrmg 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 Ž
. the yolk-sac stages with a one-way ANOVA Minitab, State College, PA, USA . Results
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
