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ICES mar. Sei. Symp., 201: 64-69. 1995
Endogenous nutrient mobilization during egg and larval
development in two marine fishes - Atlantic menhaden and spot
Hans Jørgen Fyhn and John J. Govoni
Fyhn, H. J., and Govoni, J. J. 1995. Endogenous nutrient mobilization during egg and
larval development in two marine fishes - Atlantic menhaden and spot. - ICES mar.
Sei. Symp., 201: 64-69.
Volume measurements of yolk and oil globule, and analyses of protein and free amino
acids (FA As) of developing eggs and larvae of Atlantic menhaden (Brevoorlia tyrannus) and spot (Leiostomus xanthurus) were carried out. The ratio of oil globule to yolk
is 10 times greater in spot than in menhaden. In spot, the oil globule is resorbed after
the yolk, while this occurs simultaneously in menhaden. The total protein content in
the spot embryo/larva remains constant from spawning to after first-feeding. The pool
of F A A s of spot eggs is about half that of menhaden and their decline correlates with
that of the yolk in both species. No sparing of essential FAAs occurs during resorption.
T he amount of taurine remains constant during development. It is hypothesized that,
in spot, before tissue autolysis begins energy dissipation is based 30% on FA A s and
70% on lipids, while in menhaden the corresponding values are 65% FA A and 35%
lipids.
H. J. Fyhn: University o f Bergen, Zoological Institute, Allégaten 41, N-5007 Bergen,
Norway. [Tel: (+47) 55 213588, fax: (+47) 55 329111], J. J. Govoni: National Marine
Fisheries Service, N O A A , Beaufort Laboratory, Beaufort, North Carolina28516-9722,
USA.
Introduction
The energy metabolism of marine fish larvae has
attracted much attention, with the focus on the sub­
strates that are consumed during these early stages when
first the yolk and later ingested prey from the plankton
represent the sources of nutrients. For the larvae this
conversion marks the switch from endogenous to ex­
ogenous feeding.
This transition in larval life is difficult because it corre­
lates with high mortality in cultured larval populations.
It is general consensus that this mortality relates to
nutritional inadequacy of the endogenous yolk supply or
of the exogenous feed offered the larva at the time of
first-feeding.
O ur interest in this problem has focused on the devel­
opment of the alimentary canal (Govoni, 1980; Govoni
et al., 1986) and on free amino acids (FAAs) and their
importance as a fuel for the marine fish larvae (Fyhn,
1989,1990,1993; Finn et al., 1991,1995a, b; Rønnestad
and Fyhn, 1993; Rønnestad et al., 1992a, 1993). The
studies on FAA have concerned boreal teleosts such as
Atlantic cod (Gadus m orhua), Atlantic halibut (H ippoglossus hippoglossus), lemon sole (Microstomus kitt),
and others (Fyhn and Serigstad, 1987; Fyhn, 1989; Finn
et al., 1991; Rønnestad et al., 1992a). These fishes typi­
cally have pelagic eggs with a developmental tem pera­
ture optimum of about 5°C. The eggs lack an oil globule,
so their sole endogenous supply of nutrients is that of the
yolk.
O ur results for these fishes have shown that FA A s are
an important metabolic fuel during the egg and yolk-sac
stages. A comparative study on turbot showed that its
egg oil globule is an additional fuel for the larva after
hatching when increased energy is needed for swimming
in connection with the search for prey at first-feeding
(Rønnestad et al., 1992b).
The present study was initiated to find out whether
our findings for FA A are confined to the boreal, coldwater fishes, or whether they apply more generally, and
also include warm-water species and fishes with oil glo­
bules in the egg.
Materials and methods
Eggs and larvae of the Atlantic m enhaden (Brevoortia
tyrannus, Clupeiformes, Clupeidae) and spot (Leiosto-
i c e s mar.
Endogenous nutrient mobilizations
sd. Syrap.. 201 ( 1995)
65
Table 1. Linear regressions (Y = B0 + B 1X 1) describing yolk and oil globule volume depletion in days post-fertilization for
Atlantic m enhaden ( B . tyrannus) and spot (L. xanthurus).
Species
Cohort
Slope (B,) (d -1)
Intercept (Bu) (nla)
r2
p
Yolk
Menhaden
Spot
Spot
1
2
3
-0 .1036
-0.0461
-0 .0383
542
214
210
0.82
0.84
0.84
>0.012
>0.011
>0.011
Oil globule
Menhaden
Spot
Spot
1
2
3
-0.0004
-0 .0019
-0 .0007
3
11
6
0.90
0.87
0.79
>0.004
>0.021
> 0.001-
a (nl = 10 9litre).
m us xanthurus, Perciformes, Sciaenidae) were studied.
These fishes are common along the N orth American
eastern coast, and both are commercially im portant for
industrial and food fisheries. Both species contain an oil
globule in the yolk.
Eggs and larvae were reared at 19 ± 1°C and a salinity
of 30-35 at the Beaufort Laboratory in accordance with
a previously established technique (Hettler, 1984; Pow­
ell, 1993). Lyophilized samples were shipped to Bergen
for biochemical analyses while morphometric measure­
ments were carried out on live material in Beaufort.
A mino acid analyses were carried out using the HPLC
technique on tri-chloro acetic acid extracted samples
(triplicate samples of 50 pooled eggs or 25 pooled lar­
vae), and protein by the Lowry technique on 1 M N aO H
extracted samples after removal of the FA A fraction as
described elsewhere (Fyhn and Serigstad, 1987; Finn et
al., 1991). Eggs and larvae from single batch spawnings
were used, and the larve were not fed during the experi­
mentation.
400
In spot, the newly spawned eggs contain a relatively
large oil globule of about 7 nl and a yolk sac of about
300 nl (Fig. 1A). The yolk declines to near zero in about
4 days, while the oil globule needs about 7 days to be
resorbed. No oil globule resorption seems to occur
during the egg stage, while more than 50% of the yolk
volume was resorbed at hatching. A delayed resorption
of the oil globule compared to the yolk is typical of
marine fishes (Javila and Juario, 1987; Rønnestad et al.,
1992b, 1994). A t first-feeding, energy for swimming
activity may be derived from the remaining oil globule
alone, since the yolk reserves are then exhausted.
In the newly spawned egg of Atlantic menhaden the
oil globule is smaller and the yolk larger than in spot, and
both seem to be resorbed before first-feeding (Fig. IB).
T he rate of yolk depletion is faster than that of the oil
globule during the egg stage. O ther experience with
Spot
300:
=
£
T = 19 ± 1°C
250
-
6
200
3
o>■
150
■©
100
- 4
" 2
0
2
6
4
8
10
12
1000
Firstfeeding
Hatch
_
Atlantic
Menhaden
800 -
£
a»
S
J3
600 -
C
£O
>
Results and discussion
Firstfeeding
Hatch
350
400 -
200
-
0
2
4
6
8
10
12
Days post-fertilization
Figure 1. Volume of yolk (closed symbols) and oil globule
(open symbols) in developing eggs and larvae of spot ( L .
xanthurus, upper panel) and Atlantic m enhaden (B. tyrannus,
lower panel). The data are mean values of 10 measurements on
individual eggs or larvae.
Table 2. Volume ratios and dry weight ratios of oil globule to
yolk in newly spawned eggs of spot (L. xanthurus) and Atlantic
m enhaden (B . tyrannus).
Species
Spot
Menhaden
Volume ratio
Dry weight ratio“
1:40
1:400
1:3
1:30
a Estim ated from the data in Figure 1 using a relative water
content of 93% for the yolk.
H. J. Fyhn and J. J. G ovoni
66
i c e s m ar. s d . s y m P , 201 ( 1995)
Table 3. Mensuration estimates of the eggs of Atlantic menhaden (B . tyrannus) and spot (L. xanthurus). Values for the present
study are means of 10 measurements.
Diameter
Species
Cohort
Menhaden
M enhaden
M enhaden
Spot
Spot
Spot
1
2
3
Volume
Yolk
(mm)
Oil globule
(mm)
Yolk
(nl)
Oil globule
(nl)
1.18a
0.95b
1.19“
0.828
0.816
0.80
0.163
0.22b
860
450c
580
300
280
270c
2
6C
3
_d
_e
0.23
0.21
A uthor
Present study
H ettler, 1984
Powell, 1993
Present study
Present study
Powell and Gordy, 1980
_e
6
5C
a Calculated assuming that the yolk diam eter is 74% of egg diam eter as per H ettler (1984).
b Median values.
c Calculated with mensuration formulae employed in the present study.
d No value given.
e The presence of multiple oil globules in the eggs of this cohort precluded measurements.
Firstfeeding
Hatch
16
C
T = 19 ± 1”C
c
12-
sa>
G
Series 1
Series 2
25-
T = 19 ± 1”C
■O
Firstfeeding
Hatch
Series 1
Series 2
o
S
c
8-
0
2
4
6
8
10
12
Days post-fertilization
0
2
4
6
8
10
12
Days post-fertilization
Figure 2. Protein content of eggs and larvae of spot (L. x a n ­
thurus). The values are mean ± s.d. of three samples of 50
pooled eggs or 25 pooled larvae. W here no vertical bars are
visible the s.d. is covered by the symbol.
Figure 3. Free amino acid (FA A ) content in eggs and larvae of
spot (L . xanthurus). The values are mean ± s.d. of three
samples of 50 pooled eggs or 25 pooled larvae. W here no
vertical bars are visible the s.d. is covered by the symbol.
m enhaden (Powell, 1993; Govoni, unpublished results)
has established that the oil globule is factually absorbed
prior to the yolk, although this has not presently been
confirmed. A n early consumption of the oil globule is
unusual among marine fishes. The results for menhaden
suggest that the nutritional conditions for its larvae at
first-feeding are critical for survival, because almost no
endogenous reserves are left at this stage.
The slopes of the regression lines fitted to the resorbtion data indicate that in both species the yolk is de­
pleted at a faster rate than the oil is (Table 1). These
regressions do not agree well with similar regressions for
the Atlantic menhaden of Powell (1993), but this
disparity can be explained by the fact that Powell
included neither yolk nor oil volumes of the eggs.
Neither did he include zero volumes of the day of com­
plete depletion. His work therefore describes yolk and
oil depletion solely for yolk-sac larvae; our data relate to
both the egg stage and the yolk-sac stage.
The volume ratio between the oil globule and yolk is
initially about 1:40 in spot and about 1:400 in Atlantic
m enhaden. Since the yolk has a water content of about
93% while the lipids of the oil globule exclude water, the
dry weight ratio between the two structures will be about
1:3 for spot and 1:30 for menhaden (Table 2).
Mensuration estimates compare reasonably well with
values reported in the literature for these species (Table
3).
In spot (Fig. 2), we found a constant protein content
during the egg and yolk-sac development (regrading the
Endogenous nutrient mobilizations
IC ES m ar. Sei. Symp.. 201 (1995)
4
80
F irst­
feeding
Hatch
70
60
T3
C
50
O
40
E
Hatch
—• — Series 1
— O— Series 2
F irst­
feeding
thr
val
3
leu
T = 19 ± 1”C
"O
arg
C
"c
E
2
c
s
30
s
20
tb
1
10
0
0
0
2
4
6
8
10
10
12
6
Days post-fertilization
Hatch
Figure 4. Free amino acid (FA A ) content in eggs and larvae of
Atlantic m enhaden (B. tyrannus). T he values are mean ± s.d.
of three samples of 50 pooled eggs or 25 pooled larvae. Where
no vertical bars are visible the s.d. is covered by the symbol.
decrease at hatching for series 2, see below). The protein
content decreased after first-feeding, probably related
to tissue autolysis as found for other fish larvae (Fyhn
and Serigstad, 1987; Fyhn, 1989; Rønnestad et al.,
1992a, b).
The decrease in protein content of series 2 at hatching
is due to removal of the chorion. Surprisingly, chorion
removal did not show in series 1, although the samples
were treated identically in both (i.e. protein was solubi­
lized by extracting in 1 M N aO H ). The reason for this
discrepancy between the two series is not known,
although the finding is not unique. Previously, we have
obtained similar results between different batches of
eggs from the same species (e.g. Atlantic halibut, Fyhn,
1989; Finn etal., 1991).
The total FAA pool of spot eggs was about 30 nmol/
egg at spawning, decreasing steeply in a sigmoidal pat­
tern to a low level after a latency period of about 1 d (Fig.
3). G ood agreement was obtained between the samples
from the two series. The decrease correlated with the
decrease of the yolk volume, except for the initial delay
period, which may not have been revealed in the yolk
because of the lack of sampling points. At first-feeding,
the FA A pool had attained a low level. Possibly the low
concentration of some of the F A A is the stimulus for the
larvae to feel starved and initiate feeding behaviour by
increasing swimming activity in search of exogenous
food.
In m enhaden, the egg FA A pool at spawning was
about twice as large as in spot, and the decrease was
almost linear with time (Fig. 4). A t the time of firstfeeding, the pool was much reduced.
No sparing of essential compared to non-essential
12
Firstfeedin g
tau
ser
pro
5
•a
s
4
'o
3
E
ala
e
2
1
0
8
10
12
Days post-fertilization
Figure 5. Essential (upper panel) and non-essential (lower
panel) amino acids in the free pool of eggs and larvae of spot (L.
xanthurus). The values are mean ± s.d. of three samples of 50
pooled eggs or 25 pooled larvae. W here no vertical bars are
visible the s.d. is covered by the symbol. F A A = free amino
acids.
amino acids was observed during development as the
FAA were consumed. This applied to spot (Fig. 5) as
well as to menhaden (Fig. 6).
As found typically in other fish species (Fyhn, 1989,
1993; Rønnestad et a i , 1992a, b), the amount of amino
acid analogue taurine remained constant during devel­
opment, becoming the dominant compound in the FAA
pool after first-feeding (Figs. 5, 6).
Based on our previous experience with eggs and lar­
vae of the boreal fishes, and these proximate analyses of
spot and menhaden larvae, we carried out some calcu­
lations to estimate the metabolic rate and the pro­
portional use of amino acids versus lipids as fuels during
the early life of these two warm-water fishes. For this we
assumed that there is no net synthesis of body proteins,
as was actually shown for spot (Fig. 2). Further, we
assumed that the FAA s are not plainly lost by diffusive
H. J. Fvhn and J. J. Govoni
68
ICES m ar. Sei. Sym p., 201 (1995)
10
H atch
First-
8
-O
c
Hatch
-•-th r
• val
— met
— ile
—O—leu
—o lys
-O -a rg
feed in g
6
H Lipid
c
19 ± 1 C
"O
c
o
E
~5
E
Firstfeeding
£
N
4
£
0
D ays post-fertilization
10
Hatch
Firstfeed in g
12
tau
ser
pro
Figure 8. Estim ated metabolic rate and proportional use of
various metabolic fuels in eggs and larvae of Atlantic m enhaden
(B . tyrannus). V 0 2 = oxygen uptake rate; F A A = free amino
acids.
gjy
ala
TJ'
C
o
E
e
Days post-fertilization
Figure 6. Essential (upper panel) and non-essential (lower
panel) amino acids in the free pool of eggs and larvae of
Atlantic m enhaden ( B . tyrannus). The values are mean ± s.d.
of three samples of 50 pooled eggs or 25 pooled larvae. Where
no vertical bars are visible the s.d. is covered by the symbol.
F A A = free amino acids.
Hatch
Firstfeeding
■
FA A
ED L ip id
P ro te in
T = 19 ± 1°C
Days post-fertilization
Figure 7. Estimated metabolic rate and proportional use of
various metabolic fuels in eggs and larvae of spot (L . xanthur­
us). V 0 2 = oxygen uptake rate; F A A = free amino acids.
efflux to the ambient sea water. This is in agreement with
recent measurements of larvae of the Atlantic halibut by
Rønnestad (1993). Finally, we assumed that the lipid
content of the oil globule is consumed in the aerobic
energy dissipation of the developing larvae. Although
this is not proven, consumption of the oil globule in
turbot correlates with its oxygen uptake rate (Fønnestad
et a l., 1992a).
Given these assumptions, we can calculate the
amount of oxygen dem anded to catabolize the actual
fuels, and to make a plot for the resulting oxygen uptake
during development. For spot, the estimated hourly
oxygen uptake rate at 19 ± 1°C follows a curve with a
peak of 8-10 nmol in d-1 h -1 around first-feeding (Fig.
7). Probably, the real curve, which is still to be estab­
lished by measuremënts, will be similar to this calculated
curve. Early in development F A A may account for
100% of energy dissipation, while lipids from the oil
globule may be the main fuel at first-feeding. Amino
acids derived from body proteins seem to become an
additional fuel when tissue autolysis starts. The domi­
nance of lipids at first-feeding leads to speculation as to
the actual cellular fuel during this period of develop­
ment.
Totally, FA A s seem to account for about 25% of the
energy production of the developing spot larva, while
lipids derived from the oil globule account for 60% , and
body proteins for an additional 15%. Restricting the
period of calculation to that before tissue autolysis sets
in , the proportional use of F A A and lipids as fuels would
be about 30% and 70% , respectively.
Although no data on protein are currently available
for embryonic and larval m enhaden, the same calcu­
lation procedure and assumptions as for spot reveal a
more rounded oxygen uptake curve with a peak value of
about 5 nmol i n d -1 h -1 at first-feeding (Fig. 8). FAA s
ICES mar. Sei. Symp., 201 (1995)
seem to be the dominating fuel, and account for about
65% of the total dissipated energy. Lipids from the oil
globule add to the FAA fuel, especially around firstfeeding, as in the spot. It is likely that the use of lipids for
swimming activity associated with first-feeding is a
general phenomenon in fish larvae.
Conclusions
Spot embryos contain a larger oil globule than menha­
den, and seem to rely more on lipids than on FA A in its
early life energy dissipation. Lipids seem especially im­
portant around the time of first-feeding, possibly as a
fuel for swimming activity.
Using proximate analyses as a basis for an hypothesis,
one can estimate oxygen uptake rate of larval spot and
menhaden reaching levels of about 5-10 n m o l ind“ 1I T 1
at 19 ± 1°C. These are not unrealistic values for fish
larvae at this temperature. During development and
before tissue autolysis begins, energy dissipation seems
based 30% on FA A and 70% on lipids in spot, while in
menhaden the corresponding values are 65% FAA and
35% lipids. These hypothetical values need verification
by measurements.
O ur results indicate that FA A s are likely to be an
im portant metabolic fuel during the early life stages also
for warm-water fishes, although lipids become the domi­
nant fuel after hatching in species with a high ratio of oil
globule to yolk volume. A recent study on the warmwater gilthead sea bream, Sparus aurata (Rønnestad et
al., 1994), confirms this suggestion.
Acknow ledgem ents
H JF acknowledges the extensive cooperative relations
enjoyed with the research institutions of Pivers Island,
Beaufort, USA, inspiring discussions with Ivar R ønnes­
tad and Roderick Nigel Finn, as well as excellent techni­
cal assistance provided by Maria Sula Evjen. William
H ettler provided laboratory spawned eggs. The study
was partially supported by the Norwegian Fisheries
Research Council and the Nansen Foundation.
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Endogenous nutrient mobilizations
69
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