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 Ž
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
Ž .
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 Ž
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
Ž .
Ž .
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
Ž .
opened but before the yolk-sac is completely resorbed Watanabe and Kiron, 1994 . In Ž
. 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 dd s water 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
Ž first feeding. Studies based on histomorphology of the digestive system Kjørsvik and
. 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
