Aquaculture, 80 (1989) 111-120

111
Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
First Feeding of Marine Fish Larvae: Are Free
Amino Acids the Source of Energy?
HANS JijRGEN FYHN
Zoological Laboratory, University of Bergen N-5000 Bergen (Norway)
(Accepted 29 October 1988)
ABSTRACT
Fyhn, H.J., 1989. First feeding of marine fish larvae: are free amino acids the source of energy?
Aquaculture, 80: 111-120.
Mass mortality of marine fish larvae in the ocean or in culture correlates with final yolk ab-
sorption when the larvae convert to exogenous feeding. Low planktonic abundance of appropriate
prey organisms, or lack of suitable feed, is assumed to be responsible for the mortality but criteria
to ascertain the suitability of the feed are not available. From studies of developing eggs and larvae
of halibut and cod, it is proposed that free amino acids (FAA) are an important energy source
during embryonic development of marine fishes. An exogenous supply of FAA seems necessary
when the reserves of the larva are depleted. Which specific FAA are required, and for how long,
may vary between species. This novel idea may be the clue to evaluate the suitability of a given
feed or prey organism for marine fish larvae at first feeding.
INTRODUCTION
Pelagic eggs and larvae of marine fishes develop while drifting in the water
column. Necessary nutrients to support cellular growth and energy production
during embryonic development must be contained within the egg in sufficient
quantity to satisfy the needs of the larvae until it starts exogenous feeding by
preying upon planktonic organisms. Which specific organism(s) are preyed
upon by a fish larva at this point of development remain unknown, although
it is found that larvae of marine invertebrates and other small zooplankters
are generally exploited (Hunter, 1981). Lack of appropriate food organisms
when the larva has consumed its yolk reserves will result in body tissue auto-
lysis and eventual death (Theilacker, 1981; Bagarinao, 1986). Abundance of
suitable planktonic prey organisms when fish larvae convert to exogenous
feeding has long been hypothesized as a determinant of recruitment of marine
fish populations (Hjort, 1914)) and the testing of this hypothesis still contin-
ues (Houde, 1987; Lasker, 1987). The mass mortality often experienced in
0044-8486/89/$03.50 0 1989 Elsevier Science Publishers B.V.
112
mariculture at the end of the yolksac stage is assumed to result from nutrition-
ally inadequate introductory feed (Jones and Houde, 1986).
Marine invertebrates characteristically contain high intracellular concen-
trations of free amino acids (FAA) although the composition of the FAA pool
may vary between species (Gilles, 1979). The phenomenon has been found in
all phyla of marine invertebrates (Yancey et al., 1982 ) including their egg and
larval stages (Costlow and Sastry, 1966; Barnes and Blackstock, 1975; Kas-
schau, 1975; Tucker and Costlow, 1975), as well as in marine ciliates (Kane-
shiro et al., 1969) and bacteria (Measures, 1975). Typically, FAA concentra-
tions of 200-500 mA4 are found in the tissue cells of marine invertebrates
(Yancey et al., 1982). At these high concentrations, FAA participate in cellular
osmoregulation by counteracting the dehydrating effect of the high ambient
salinity of the sea water (Fyhn, 1976; Pierce, 1981). Since invertebrate larvae,
particularly copepod nauplii, are a major component of the early diet of marine
fish larvae (e.g. Last, 1978; Checkley, 1982), the latter are guaranteed a high
intake of FAA when they convert to exogenous feeding. This fact has so far not
been considered in nutritional studies of first-feeding marine fish larvae. Re-
search efforts in this field currently focus on lipids and proteins, together with
the indispensable nature of the nutrients (Watanabe et al., 1983; Cowey et al.,
1985 ). Surprisingly little attention is given to another aspect of a diet, namely,
its ability to provide energy by aerobic catabolism. To support growth and
survival of a developing fish larva, anabolic precursors as well as energy for
synthesis, maintenance, and homeostasis must be provided. Based on the fol-
lowing results and considerations, I propose that FAA are an important sub-
strate for energy production during embyrogenesis and early larval develop-
ment in marine fishes.
MATERIALS AND METHODS
Eggs and yolksac larvae of the Atlantic halibut, Hippoglossus hippoglossus,
and cod, Gudus morhua, were reared in flow-through aquaria at 7? 1 and
5 ?0.2C, respectively (Senstad, 1984; Solberg and Tilseth, 1986). Egg and
larval samples were taken regularly during development, extracted in 6% tri-
chloroacetic acid (TCA), and analysed for FAA and total protein. Pooled sam-
ples of cod eggs and larvae were treated as described by Fyhn and Serigstad
(1987). Halibut larvae were individually extracted in 1 ml of TCA and the
supernatant used for analyses of FAA while the precipitated larval bodies were
taken for protein determination. The supernatant was diluted (1: 25) in ultra-
pure 0.024 M HCl and analysed on an automatic amino acid analyser (Chro-
maspek J 180, Hilger Analytical) with fluorimetric detection (o-phthaldialde-
hyde reagent) and high pressure loading. Amino acid standards (1 nmole)
were included for every eighth sample and 6% TCA, appropriately diluted in
the HCl, was used as a blank. The TCA in the applied concentrations did not
113
interfere with amino acid separation or quantification. The analytical repro-
ducibility varied between 2 and 10% for the various amino acids.
Protein content was determined by the method of Lowry et al. (1951) using
the modification for micro-quantities by Rutter (1967). The precipitated lar-
val bodies were rinsed in distilled water and dissolved in 0.5 M NaOH over-
night. Crystalline bovine serum albumin (BSA) dissolved in 0.5 M NaOH was
used as standard, and 0.5 M NaOH as a blank. The BSA was calibrated in 0.9%
NaCl solution at 280 nm using an absorption coefficient of E:tm =6.15. The
samples were read in triplicate and the reproducibility was better than 5%.
RESULTS AND DISCUSSION
The total contents of FAA and body protein in developing halibut embryos
are shown in Fig. 1. About 2 pmoles of FAA were present in the embryo at
hatching but about 1.7 pmoles were depleted during the subsequent 3 weeks.
The depletion occurred without a simultaneous increase in protein content,
indicating that the FAA were not removed by net protein synthesis. If polym-
2.5 -
.
;;
2.0 -
L
L?
\
S
E 1.5-
I
800
0.0 1
0
I I 1
IO 20 30 40 50
Days after fertilization
Fig. 1. Content of free amino acids and body protein in developing larvae of Atlantic halibut from
hatching to final yolk absorption. The data (mean f s.d.) refer to analyses of N individual larvae
as given.
114
erized, the depleted amount of FAA would correspond to about 182 pg of pro-
tein (calculated from the various FAA involved (see Fig. 2)) their molecular
weights, and correcting for 1 mole of water excluded per mole of peptide bonds
formed). The total content of FAA in the halibut embryo at hatching equalled
about 30% of the amount of amino acids contained in its body proteins.
The changes occurring in individual FAA of developing halibut embryos are
shown in Fig. 2. Most of the FAA decreased steeply during the first 3 weeks
after hatching and then leveled off or declined slowly over the following 4 weeks.
Taurine deviated from this pattern and showed no major changes during the
recorded 7 weeks of embryonic development. Serine, glutamic acid, alanine,
leucine, and lysine accounted together for about 60% of the decrease in the
Fig. 2. Changes in content of individual free amino acids in developing larvae of Atlantic halibut.
Hatching marked by stippled line. The data (mean ? s.d.) refer to analyses of N individual larvae
as given for data on amino acid content in Fig. 1.
115
FAA pool during the first 3 weeks after hatching. When valine, threonine, as-
partic acid, arginine, and isoleucine were also considered, almost 90% of the
decrease was accounted for. Essential amino acids (arginine, histidine, isoleu-
tine, leucine, lysine, methionine, phenylalanine, threonine, tyrosine, and va-
line; Walton, 1985) and non-essential amino acids contributed about equally
to the decrease in the FAA pool (47 and 53%, respectively).
If the depleted 1.7 pmoles of FAA (corresponding to 182 pg of protein as
calculated above) were removed by aerobic catabolism, a total of 191 ~1 of
oxygen was needed as estimated by applying a metabolic value of 1.05 1 oxygen/
g protein as recommended by Brett and Groves (1979) for catabolism of body
proteins in fish. Oxygen uptake rates of halibut larvae, taken from the same
batch as was sampled and analysed for FAA, have been measured at 5 C (Ser-
igstad, 1987). From those data, a total oxygen consumption of 180 ~1 was es-
timated for a halibut larva over the first 3 weeks after hatching. At 7C, the
mean rearing temperature, the oxygen consumption of the halibut larvae would
be increased due to a temperature effect on the metabolic rate. In a recent
review, Rombough ( 1988) found literature data for the temperature effect ( Qlo
values) on oxygen uptake rates of fish embryos and larvae to be highly variable,
ranging from 1.5 to 4.9 with an average of about 3.0. Using this average value,
and the data of Serigstad (1987), the total oxygen consumption of a halibut
larva at the rearing temperature can be estimated to be about 225 ~1 over the
first 3 weeks after hatching.
Similar studies of cod eggs and yolksac larvae (Fyhn and Serigstad, 1987)
have shown a FAA content of about 0.2 pmoles in the egg at spawning, and a
depletion of about 0.18 pmoles during the period from spawning to day 5 after
hatching. Alanine, serine, leucine, isoleucine, lysine, and valine, in that order,
quantitatively dominated the FAA pool, and together accounted for about 75%
of the decrease. The FAA pool was found to decrease without a corresponding
increase in the body protein content. A stable protein content of cod embryos
during this developmental period agrees with previous findings (Buckley, 1981) .
As in the halibut larva (Fig. 2)) taurine was found to remain at a constant level
during embryonic development in the cod (Fyhn and Serigstad, 1987). The
total content of FAA in the cod egg at spawning equalled more than 50% of the
amount of amino acids contained in its body proteins. The oxygen consump-
tion of a developing cod embryo from spawning to day 5 post-hatch (measured
at the rearing temperature, 5C), amounted to about 16 ~1 (Fyhn and Serig-
stad, 1987). The ammonia excretion of developing cod embryos (5C) has
been measured by Davenport et al. (1983). From their plotted data, a total
excretion of about 0.27 pmoles ammonia/embryo can be estimated for the pe-
riod from spawning to day 5 after hatching.
Good agreement was found (Table 1) when the actual oxygen consumption
of halibut and cod embryos was compared with the amounts of oxygen neces-
sary to catabolize the FAA that actually disappeared during the given devel-
116
TABLE 1
Comparison between measured and calculated values of oxygen consumption and ammonia ex-
cretion in developing embryos of Atlantic halibut ( 7 + lo C ) and cod (5 ? 0.2 C )
Species
Halibut
Cod
Developmental Decrease in Oxygen Ammonia
period FAA content consumption, excretion,
(,amoles/embryo) measured measured
calculated calculated
( Nembw )
(pmoles/embryo )
Hatching to 1.7 225 (no data)
day 21 post-hatch
191
Spawning to 0.18 16 0.27
day 5 post-hatch
19 0.25
The calculated values refer to an oxidative catabolism of the free amino acids (FAA) actually
disappearing during the tested developmental periods (halibut: Fig. 1, cod: Fyhn and Serigstad,
1987). The measured oxygen consumption of halibut larvae at 5C (Serigstad, 1987) has been
adjusted to the rearing temperature by applying a Q1,, of 3.0 (Rombough, 1988). The measured
ammonia excretion of cod embryos was taken from Davenport et al. (1983).
opmental periods. In addition, for cod embryos, the amount of ammonia ex-
pected from this catabolism compared well with the actual ammonia excretion.
The amount of ammonia expected from FAA catabolism was estimated by stoi-
chiometry, taking into account the extra amino groups of arginine, histidine,
and lysine, and an ammonia store of about 0.02 pmoles which was released by
the cod egg at hatching (Fyhn and Serigstad, 1987). The close agreement be-
tween the calculated and measured oxygen consumption (Table 1) leaves little
room for other nutrients than FAA as fuel for aerobic energy production in
halibut and cod embryos. Oil globules are not present in these embryos so there
are minimal reserves of triacyl lipids available as a possible fuel. The agree-
ment between the calculated and measured ammonia excretion, in conjunction
with the maintained body protein content during embryonic development, fur-
ther points to FAA as the actual fuel for energy production in halibut and cod
embryos.
In this comparison it is recognized that halibut and cod have quite different
egg and larval characteristics. For example, a halibut egg is about 15 times
heavier than and contains about 10 times the FAA content of a cod egg. A
halibut embryo hatches in quite an immature state compared to the cod but,
conversely, has a yolksac stage lasting about 50 days at 5C (Blaxter et al.,
1983 ) compared to about 7 days for the cod at this temperature (Tilseth et al.,
1984 ) . Moreover, halibut larvae appear ready to begin exogenous feeding 28
35 days after hatching when reared at 5C (Blaxter et al., 1983), while cod
larvae start exogenous feeding 5-6 days after hatching at this temperature
(Tilseth et al., 1984). In spite of these embryonic differences, however, a re-
117
markable agreement was found between the calculated and measured meta-
bolic values for both species (Table 1) .
The data support the idea that FAA are the main substrate for aerobic energy
production during embryogenesis of halibut and cod. Probably this applies to
other marine fishes as well, because their eggs, where tested, contain large
amounts of FAA (Suzuki and Suyama, 1983). The data base needs to be ex-
tended. A dependence of fish embryos on FAA is consistent with the widely
held view that adult fishes derive a large fraction of their metabolic energy
from proteins (i.e. amino acids) whether they are fed or starved (Tacon and
Cowey, 1985; Walton, 1985). To support maximum growth rate, young fishes
demand twice the proportion of dietary protein as compared to the require-
ments of young homeothermic vertebrates (Bowen, 1987).
Marine fish larvae, especially those hatching from pelagic eggs, may require
a supply of FAA after the depletion of their own reserves, because the digestive
tract is morphologically and functionally incomplete when they initiate exog-
enous feeding (Tanaka, 1969, 1973; Blaxter et al., 1983; Govoni et al., 1986;
Ferraris et al., 1987). A low proteolytic capacity of the intestine during this
period is suggested by the absence of aminopeptidase in the intestinal mucosa
of larvae of the marine milkfish, Churws chunos, during the first l-2 weeks
after the start of exogenous feeding (Ferraris et al., 1987), and by the low
trypsin content in larvae of herring, Clupea hmengus, during the same period
(Pedersen et al., 1987). Until the intestine is adequately differentiated, the
FAA from ingested food could provide an indispensable source of energy. In
this connection it seems pertinent to recall that, in nature, a large supply of
FAA is ingested when marine fish larvae prey upon invertebrate larvae, roti-
fers, ciliates or other small zooplankters during first feeding. The nutritional
value of these FAA needs to be considered. Surprisingly, assimilation of FAA
from ingested food was not dealt with by Govoni et al. (1986) in their review
of the physiology of digestion in fish larvae.
Marine fish larvae are selective feeders both for type and size of prey (e.g.
Last, 1978). This selectivity may imply ingestion of different pools of FAA
depending upon the type of prey caught, and result in a match between the
FAA pool of the preferred prey and the FAA pool that was consumed during
endogenous feeding in the yolksac stage. It is conceivable that such a match in
FAA pools is optimal for continued energy production in the fish larva during
the critical period when it converts from endogenous to exogenous feeding.
Thus, prey selectivity may play an important role during early larval
development.
Following this line of thought, a guiding principle can be offered to help
identify the right natural prey organisms of a fish larva at first feeding,
namely, that the FAA pool of the prey should match the FAA pool consumed
by the fish embryo during endogenous feeding. Similarly, when preparing an
artificial first feed of a fish larva in mariculture, I suggest that it would be
118
beneficial for larval growth and survival to include in the feed - which needs
to be appropriately encapsulated - a pool of FAA matching that consumed by
the fish embryo during endogenous feeding.
CONCLUSIONS
The available data and related considerations point to FAA as a main source
of energy during embryogenesis of marine fishes. This may continue to apply
for some time beyond depletion of its own FAA reserves when the larva starts
exogenous feeding. Which specific FAA are required, and for how long, may
vary between fish species. The hypothesis has strong implications for the for-
mulation of an artificial first feed for marine fish larvae and should facilitate
the identification of the natural prey organisms ingested at this early stage of
development.
ACKNOWLEDGEMENTS
Halibut larvae were provided by Knut Senstad and Ingvar Huse, manager,
Austevoll Aquaculture Station, and cod larvae were provided by Tor Solberg,
Institute of Marine Research, Bergen. Analytical assistance of Lutz Hahnen-
kamp, Maria Sula Evjen, and Torunn Ellingsen, and support received from the
Norwegian Research Council for Science and the Humanities are acknowl-
edged. I greatly appreciated discussions and critical comments by Drs. Don F.
Alderdice, Trevor P.T. Evelyn, and Jan N.C. Whyte at the Pacific Biological
Station, Department of Fisheries and Oceans, Nanaimo, Canada during prep-
aration of the manuscript.
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