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 Artemia enzymes
200 Artemia Digestive system
Trypsin 52.66.7
4.2 50.3
8.4 Amylase
5,44930 435.9
833.1 52.3
Lipase 6.30.7
0.5 19.0
2.6 Alkaline phosphatase
68.89.2 5.5
57.1 9.6
a
Ž Means1 SE
ns 3 pooled samples of ca. 14,000 Artemia nauplii or ns 3 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 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
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
. Bailey, 1995 , suggests limited ability of Atlantic halibut yolk-sac larvae to digest
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 58C, 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 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 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
Ž .
Ž Cousin et al., 1987 or the feeding of an inadequate diet Cahu and Zambonino Infante,
. 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
. of Atlantic halibut was relatively small 2–10 , and the contribution to trypsin and
lipase levels we recorded are similar to those found in walleye pollock larvae fed rotifers Ž
. Ž
Oozeki and Bailey, 1995 and in striped bass larvae fed Artemia nauplii Ozkizilcik et .
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
