RNA:DNA ratios and growth of herring (Marine Biology (1996) 126:591 602
9 Springer-Verlag 1996
A. F o l k v o r d 9 L. Y s t a n e s 9 A. J o h a n n e s s e n
E. M o k s n e s s
RNA: DNA ratios and growth of herring (Ciupea harengus) larvae reared
in mesocosms
Received: 28 March 1996/Accepted: 31 May 1996
Abstract Autumn-spawned North Sea herring larvae
(Clupea harengus L.) were released in two outdoor
mesocosms of 2500 m 3 (A) and 4000 m 3 (B). The mesocosms were monitored for temperature, salinity, oxygen,
chlorophyll a, zooplankton and herring larvae abundance. The density of suitable prey for first feeding
larvae (mainly copepod nauplii) was initially low in
Mesocosm A ( < 0.1 1-1) compared to in Mesocosm
B ( > 1 1-1). Half-way through the experiment the situation was reversed, with higher densities of prey in
Mesocosm A ( > 31 1) as compared to Mesocosm
B ( ~ 1 1-1). The average temperature declined steadily
in both mesocosms from 18 ~ at release to 11-12 ~ by
the end of the experiment 60 d later. The RNA: DNA
values of individual herring larvae were related to
protein growth rates and temperature adjusted according to Buckley (1984). A corresponding DNA growth
index (Gdi) was given as: Gdi = 0.68 T E M P + 3.05
RNA : DNA - 9.92. The RNA : DNA based growth indices were significantly correlated with other somatic
growth estimates. The average estimated protein growth
rate in the two mesocosms followed the same temporal
pattern as the somatic growth rate, but with a lag of
2 d or more. Residual analysis of the regression of In
RNA versus In DNA also showed the same temporal
pattern as the RNA:DNA ratios, but the shift in condi-
Communicated by L. Hagerman, Helsingor
A. Folkvord ( ~ ) . L. Ystanes 1 9 A. Johannessen
Department of Fisheries and Marine Biology,
University of Bergen, Bergen High Technology Centre,
N-5020 Bergen, Norway
E. Moksness
Institute of Marine Research, Flodevigen Marine
Research Station, N-4817 His, Norway
Present address:
1Dyno Oil Field Chemicals, P. O. Box 2448,
N-5037 Solheimsviken, Norway
tion as estimated by this method occurred more in
synchrony with the other somatic growth measures.
Larvae in Mesocosm A had RNA : DNA values similar
to the starvation control kept in the laboratory the first
days after release, confirming that larvae in Mesocosm
A initially were in poor nutritional condition. On the
other hand, the majority of the herring from Mesocosm
B were characterised as starving or in poor nutritional
condition towards the end of the experiment. The assessment of growth and nutritional condition were in
accordance with independent survival estimates which
suggested that the majority of the total mortality occurred during the first 15 d in Mesocosm A and thereafter in Mesocosm B.
Introduction
The nutritional condition of the larval stages has long
been considered to be of major importance for subsequent survival and recruitment in fishes (Hjort 1914).
A poor nutritional condition can directly lead to increased mortality by starvation or indirectly through
prolonged stage duration and predation (Ehrlich et al.
1976; Shepherd and Cushing 1980; Booman et al. 1991).
Many hypotheses regarding the important factors
regulating survival during the earliest periods of life
rely on adequate methodology to assess the nutritional
condition or growth of larval fish. Several morphometric and other measures have thus been used to assess
larval condition, and to infer recent growth (Ferron
and Leggett 1994). The use of nucleic acids is a relatively recent methodology used for this purpose (Buckley
1984), and investigations have been conducted to investigate the validity of RNA : DNA ratios as an index of
larval condition or growth (Clemmesen 1994; Westerman and Holt 1994).
Several studies have been conducted with Atlantic
herring, Clupea harengus, in the laboratory, and
592
conclusions from these studies regarding the appropriateness of RNA:DNA ratios have been somewhat
contradictory (e.g. Clemmesen 1994; Mathers et al.
1994). A serious limitation of these laboratory studies
has been that the larval groups have experienced rather
poor growth and/or high mortality in spite of excess
feeding (Folkvord and Moksness 1995). This suggests
that factors other than food availability are influencing
the results of laboratory studies, and caution must
therefore be taken when interpreting the results. We
decided to use large outdoor mesocosms to rear herring
larvae under semi-natural conditions. This approach
has previously been used with success to obtain larval
and otolith growth data in herring (Moksness and
Wespestad 1989; Moksness 1992). The advantages of
this approach are that large numbers of known-age
larvae can be sampled under well-described conditions
(Oiestad 1990), and that herring larvae exhibit relatively high growth rates at plankton densities comparable
to those in the field (Oiestad and Moksness 1981; Fossum and Moksness 1995).
Two mesocosms with contrasting initial feeding conditions were set up with herring larvae to investigate
the effects on observed somatic growth rates and
estimated growth rates from RNA:DNA ratios. We
established relatively good feeding conditions in one
mesocosm, and marginal feeding conditions in the
other. Large numbers of larvae were sampled throughout a 60-d period to determine size- and growthdependent effects on RNA:DNA ratios and other
growth indices.
Materials and methods
Eggs from Buchan North Sea herring (Clupea harengus L. ) were
fertilised in the laboratory on 23 August 1991. The eggs were
incubated at 13~ (_+ 0.2) and 32~o salinity. About 18 000 larvae
hatched on 1 September constituted Group 1 and were released on
2 September in Mesocosm B. Group 2 hatched 2 September, and
5000 larvae were released on 3 September in Mesocosm A. A third
group of 500 larvae, also hatched on 1 September, were kept in the
laboratory at 16.5~ in five 8-1itre cylinders with 100 larvae each as
starvation controls. Survival was estimated by terminating the experiment and counting the number of larvae in one cylinder every
other day. Subsamples of about ten larvae from each cylinder were
used in the R N A : D N A analysis. Both mesocosms are located at
Fodevigen Research Station, Norway. Mesocosm A is 4 m deep and
has a volume of 2500 m 3, while Mesocosm B was initially 2 m deep
and around 3 m deep at the end of the experiment. The volume
during this period increased from 2500 to 4000 m 3 mainly due to
added sea water and also due to some precipitation. The main
experiment lasted until Day 65 to 67 after hatching, when the
mesocosms were drained and the remaining herring larvae and
juveniles were collected. Between 100 and 200 live juvenile herring
(age 65 to 67 d) were transferred from each of the mesocosms to
holding tanks in the laboratory. These herring were kept at 12~
and were not offered food. About ten fish from each group were
sampled after 0, 3 and 6 d (Group 1) and 7 d (Group 2) of starvation.
Temperature was monitored daily during the first half of the
experiment, and about every second day thereafter. It was measured
at 0.5 m depth and at 1 m depth intervals from surface to bottom.
Salinity and oxygen were monitored at the same depths about once
a week. Average chlorophyll a content in the upper 2 m was measured twice weekly. Microzooplankton was sampled in 100-1itre
portions approximately twice weekly with a 300 litre rain-1 capacity pump from the same depths as the hydrography series. Macrozooplankton and herring larvae were collected with diagonal hauls
taken around midnight with a 0.3 m 2 two-chambered net with
500 lain mesh size. Larvae from one chamber were used for
R N A : D N A analyses, and larvae from the other chamber were
fixated in 96% ethanol for morphometric and otolith analyses
(Moksness et al. 1995). Further details regarding sampling and
localities are given in Wespestad and Moksness (1990) and Rokeby
(1991).
Morphometric and biochemical analyses
Herring larvae used in the R N A : D N A analyses were measured
(standard length, SL) alive after capture and subsequently frozen in
liquid nitrogen. Only larvae older than 2 wk were generally alive
after capture and suitable for live measurements. Larvae fixated in
96% ethanol were measured to the nearest 0.1 mm under a dissecting microscope 2 mo after sampling, in the same manner as the live
larvae, and dry weight was measured (60 ~ minimum 24 h) on
a Sartorius microbalance to the nearest 1 lag. The larval sizes were
not corrected for shrinkage (Moksness et al. 1995).
All chemicals used in the RNA:DNA analyses were analytical
grade from Sigma Chemical Co.: DNA from herring sperm (Cat. No.
9007.4.2), RNA from yeast (Cat. No. 63231.63.0), RNAase from
cattle pancreas (Cat. No. 9001.99.4), and ethidium bromide (EB, Cat.
No. 1239.45.8). The methodology described in Raae et al. (1988) was
used with slight modifications. The larvae were frozen individually
in 1.5-ml Eppendorf tubes with as little water as possible. Prior to
analysis, a drop of ice-cold Tris-EDTA buffer (0.05 M Tris, 0.1 M
NaC1, 0.01 M EDTA, adjusted to pH 8.0 with HC1) was added to the
larva. The larva was disintegrated by applying a short pulse of
ultrasound (Virtis 50, 25 W). After adding 0.5 ml of the same buffer,
the larva was completely homogenised by two separate 10-s pulses
of ultrasound. After homogenisation the material was centrifuged at
9500 x g and - 2~ for 15 min. Total nucleic acid concentration
(RNA + DNA) was determined fluorometrically with a PerkinElmer LS-5 (excitation: 360 nm, emission: 590 nm) by adding 2.8 ml
EB-buffer solution (5 gg EB ml- ~ buffer) to a 200 ~tl aliquot of the
supernatant. DNA concentrations were determined in the same way
after incubation of another 200 gl aliquot with 5 lag RNAase for
30 min at 37~ The fluorescence of RNA was adjusted according to
Le Pecq and Paoletti (1966). Total DNA content was estimated by
means of a calibrated D N A standard curve. The larvae collected at
the termination of the experiment contained too much skin and
skeletal material to be completely homogenised with the ultrasound,
and only muscle filets from these individuals were used.
R N A : D N A ratios were converted to temperature-adjusted protein growth rates (Gpi) by applying the equation from Buckley
(1984):
Gpi =0.93 T + 4.75 R N A : D N A ~- 18.18,
where T is the average temperature in ~ in the respective mesocosm
on the day of sampling. Larvae with protein growth rates < 0 were
considered starving (Robinson and Ware 1988), and larvae with
protein growth rates < 2 were considered to be in poor nutritional
condition.
Statistical analyses
Age-dependent size data (SL, dry weight, otolith radius and total
DNA) were fitted with polynomial regression to obtain close fit to
the data. The time derivative of these functions was used to obtain
593
age-dependent population growth rates. The obtained growth rates
corresponding to the first and last week of available data were
excluded due to low precision of the polynomial fits at the borders.
The dry weight, otolith radius, and total DNA data were log transformed (ln DW, In OTO, In DNA) prior to the data fitting to obtain
specific growth rates. Stepwise regressions were carried out to model
growth rate as a function of R N A : D N A ratios, temperature, and
one of the size measures. Input values were the observed average
R N A : D N A and temperature values, and the corresponding estimated size and growth rates obtained from the polynomial regressions. These values were also used as input values in a principal
component analysis (PCA) describing the similarities between the
various size and growth measures.
Differences in morphological and biochemical relations of herring
larvae from Mesocosm A and Mesocosm B were tested with
ANCOVA with size (SL or in DNA) as covariate. Survival was
estimated with linear regression after In transformation of the
abundance estimates. The sample taken at the day of release was
omitted due to patchiness of larvae. Differences between groups
were considered significant at probability levels below 0.05. All
statistical analyses and data presentations were carried out with
Statistica for Windows.
Results
Hydrographical and environmental conditions
The average temperature was around 18~ in both
mesocosms at release, and it declined steadily to
10-12~ by the end of the experiment. The temperature
at 0.5 m depth was similar in both mesocosms but the
temperature at 3 m was around 2~ higher in the
deeper mesocosm (A) than in Mesocosm B (Fig. 1). The
salinity in the deeper half of the mesocosms remained
above 30Tooo,and the salinity at 0.5 m depth was above
17.5~o throughout the experiment. A pycnocline was
present between 0.5 and 1 m depth during the latter half
of the experiment in both mesocosms. Oxygen saturation was generally above 80% in the entire water column in both mesocosms except during a 2-wk period in
the deepest part in Mesocosm B where a minimum
saturation of 30% was measured.
The chlorophyll a content in Mesocosm B was
higher and more variable than in Mesocosm A and
averaged 15 and 2 gg 1-1, respectively, with no apparent trends during the experiment. The pump samples
showed that the density of copepod nauplii in Mesocosm A increased markedly from low levels at release
(< 0.1 1-1) to relatively high densities towards the end
of the experiment (> 1 1-1, Fig. 2a). Mesocosm B, on
the other hand, had higher initial densities (> 0.5 1-*),
but experienced a reduction in the latter half of the
experiment ( < 0.2 l - t , Fig. 2a). A similar trend was
observed with the other larger zooplankton organisms
sampled with the two-chambered net. Mesocosm A had
an initial density of other zooplankton organisms below 0.5 1 t, increasing up to 7 1-t towards the end of
the experiment (Fig. 2b). Mesocosm B had densities up
-+-A
0
20 84
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Age (days)
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50
60
Age (days)
Fig. 1 Clupea harengus.Temperature (~ at 0.5 m and at 3 m depth
in Mesocosms A and B during the experiment
Fig. 2 Clupeaharengus.Average densities (1- l) of (a) nauplii from
pump samples and (b) other zooplankton organisms (nauplii excluded) from net samples in Mesocosms A and B
1 Clupea harengus. Regression equations of number of herring larvae caught per haul in Mesocosms A and B up to Day 15 after
hatching. Y is number of larvae caught per haul, X is larval age in days. Numbers in parentheses are SE of respective parameters; p shows
probability that slope = 0
Table
Regression equations
Mesocosm A
Mesocosm B
In Y = 3.149 (0.224) - 0.100 (0.024) X
In Y = 5.032 (0.150) - 0.012 (0.016) X
R2
0.566
< 0.001
n
13
12
p
< 0.002
0.466
594
to 7.5 1-t during the first half of the experiment, but
they dropped to about 1 1-1 towards the end of the
experiment (Fig. 2b). In Mesocosm A the most abundant zooplankton group was calanoid copepods,
whereas harpactoid copepods dominated in Mesocosm B.
30 84
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+++;
10
+
+
Survival and size-at-age
I
The survival of larvae estimated from the two-chamber
net hauls indicated that around 25% of the larvae in
Mesocosm A were still alive 15 d after hatching. No
significant mortality was observed in Mesocosm B up
to this age (Table 1). The number of larvae caught per
haul in Mesocosm B showed a declining trend after
Day 15, but part of this reduction could be due to
avoidance. The number of herring recovered at termination of the experiment was 864 on Day 65 in Mesocosm A and 2116 at Day 67 in Mesocosm B. Adding
the 864 and 2116 larvae that were sampled from Mesocosms A and B, a total of around 27% of the larvae
flom both mesocosms were recovered during the experiment.
The size-at-age (length, dry weight and total D N A
content) data showed a different pattern in Mesocosm
A compared to in Mesocosm B. The size-at-age was
higher initially in Mesocosm B than in Mesocosm
A (Fig. 3a-c). The standard length averaged 16 mm
around Day 17 in Mesocosm B, and Day 32 in Mesocosm A (Fig. 3a). Dry weight averaged 0.2 mg
around Day 8 in Mesocosm B, and Day 25 in Mesocosm A (Fig. 3b), and the larvae contained 1 tzg of
D N A on Day 7 in Mesocosm B and Day 22 in Mesocosm A (Fig. 3c). The age of equal average size in the
two mesocosms was estimated by the intersection of the
polynomial fits of the respective size-at-age data (Fig. 3;
Table 2). The average size of the larvae in the two
mesocosms was similar around Day 40 to 44 according
to the various size measures (Table 3).
A decrease in total D N A content was observed in the
starvation control group kept in the laboratory
(Fig. 3c). The average D N A content decreased from
around 0.48 gg D N A per larva at release to 0.37 ~tg
DNA per larva on Day 11 when the starvation control
died out. The corresponding average D N A content on
Day I t in larvae from Mesocosm A and Mesocosm
B was 0.65 and 1.9 pg per larva, respectively (Fig. 3c).
The standard length of the fixated larvae used for
otolith analysis (Moksness et al. 1995) was slightly
longer than the live measured larvae used for the
R N A : D N A analysis during most of the experiment.
The difference was at most 2 mm (approximately 12%
relative difference) on any given day, and the average
larval lengths in the two mesocosms were equal 3.5 d
earlier based on the fixated larvae than based on the
live larvae (Table 3).
I
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.<1
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-= 0
_1 84
-2
0
110
20
3'0
Age (days)
410
5;
60
Fig. 3. Clupea harengus. Age-specific (a) standard length (SL, mm)
of live larvae; (b) dry weight (DW mg, in-scale) of fixated larvae
(from Moksness et ai. 1995) and (e) total D N A content (~tg, in-scale)
of individual herring larvae in Mesocosms A and B. Lines represent
polynomial fits to the data (Table 2). The regression equation of the
starvation control, C, is: In D N A = - 0.816 + 0.0445 X - 0.00564
X 2, R 2 = 0.463, n = 37, where X is age (d)
Temporal patterns in R N A ' D N A ratios
and growth estimates
The R N A : D N A ratios of larvae were below 2.0 initially, but increased rapidly to over 2.0 in Mesocosm B,
whereas it remained below 2.0 in Mesocosm A until
Day 20 (Fig. 4). In the starvation control group, the
RNA:DNA ratio dropped to below 1.0 after Day 6 to 7,
and was about 0.4 on Day 11 when the group died off
(Fig. 4). Based on the RNA:DNA values, the larvae
from the starvation control had protein growth rates
similar to the larvae from Mesocosm A until Day 5, but
continued to drop to values below 0% d-1 on Day 7.
On Day 9 and 11 the average estimated protein growth
rate was around - 1 to - 2 % d - 1
595
T a b l e 2 Clupea harengus. P o l y n o m i a l r e g r e s s i o n e q u a t i o n s d e s c r i b i n g t h e r e l a t i o n b e t w e e n l a r v a l a g e in d a y s (X) a n d m o r p h o l o g i c a l ,
b i o c h e m i c a l , a n d p r o t e i n g r o w t h (Gpi) m e a s u r e s o f h e r r i n g l a r v a e in M e s o c o s m s A a n d B. D a t a o n f i x a t e d l a r v a e (.fix) f r o m M o k s n e s s et al.
(1995)
Dependent
v a r i a b l e s (Y)
Mesocosm A
In D W (fix)
S L (fix)
In O T O (fix)
S L (live)
In D N A
RNA:DNA
Gpi
Mesocosm B
In D W (fix)
SL (fix)
in O T O
(fix)
SL (live)
In D N A
RNA:DNA
Gpi
Regression equations
Rz
n
Y
Y
Y
Y
Y
Y
Y
=
=
=
=
=
=
=
3 . 2 4 6 + 0 . 1 7 8 4 X - 1 . 8 2 4 E - 2 X 2 + 8 . 9 4 E - 4X 3 - 1 . 6 3 1 E - 5 X 4 + 1 . 0 0 4 E - 7 X s
7.67 + 0 . 5 3 8 X - 5 . 5 8 1 E - 2 X 2 + 2 . 7 8 8 E - 3 X 3 - 4 . 9 4 1 E - 5 X 4 + 2 . 8 8 7 E - 7 X 5
2.315 + 0 . 0 9 2 X - 9 . 3 5 E - 3 X 2 + 4 . 6 6 3 E - 4X 3 8 . 7 4 6 E - 6 X 4 + 5 . 5 7 1 E - 8 X s
45.26 - 4.492X + 0.200X 2 - 3.431E - 3X 3 + 2.088E - 5X 4
- 0.54 - 0 . 0 1 9 X + 4 . 1 8 E - 3 X 2 - 1 . 7 1 E - 423
q- 4 . 5 3 E -- 6 X 4 - 4 . 2 4 E - 8 X s
1.58 - 0 . 0 7 8 X + 4 . 4 7 E - 3 X 2 + 5 . 7 0 E - 5 X 3 - 3 . 4 2 E - 6 X 4 + 2 . 7 9 E - 8 X 5
7.46 - 1 . 0 5 1 X + 7 . 6 6 E
2X 2 - 2.24E - 3X 3 + 3.68E - 5X ~ - 2.75E - 7X s
0.955
0.962
0.960
0.840
0.858
0.717
0.570
i25
127
127
99
161
161
161
Y
Y
Y
Y
Y
Y
Y
=
=
=
=
=
=
=
- 3.786 + 0.4474X - 2.799E2X 2 + 1.033E - 3X 3 - 1.869E - 5X 4 + 1.260E - 7X s
5.42 + 1.138X - 3.59E - 2X 2 + 8.76E - 4X 3 - 1.57E - 5X 4 + t.21E - 7X 5
2 . 2 6 4 + 0 . 0 8 7 X - 1 . 1 3 4 E - 3X z + 4 . 9 7 5 E
5X 3 - 1.782E - 6X 4 + 1.774E - 8X s
17.34 - 0.542X + 0.0349X 2 - 4.672E - 3X ~
- 1.08 + 0 . 2 0 5 X - 8 . 4 1 E - 3 X 2 + 2 . 7 7 E - 4 X 3 - 4 . 3 4 E - 6 X 4 + 1 . 8 5 E - 8 X s
0.61 + 0 . 3 9 8 X - 2 . 4 9 E - 2 X z + 6 . 7 0 E - 4 X 3 - 8 . 2 9 E - 6 X 4 q- 3 . 7 6 E - 8 X s
4.94 + 0.594X + 3.17E - 3X 2
2.62E - 3X 3 + 8.59E - 5X 4 - 8.05E - 7X s
0.879
0.872
0.927
0.576
0.831
0.284
0.398
158
159
I59
136
208
208
208
T a b l e 3 Clupea harengus. A g e o f s i m i l a r size a n d g r o w t h r a t e s o f
l a r v a e in M e s o c o s m s A a n d B. D a t a o n f i x a t e d (fix) l a r v a e f r o m
M o k s n e s s et al. (1995). P r o t e i n g r o w t h r a t e s (Gpi) w e r e o b t a i n e d
u s i n g r e l a t i o n g i v e n b y B u c k l e y (1984)
Measure
A g e o f e q u a l size in
m e s o c o s m s (d)
Age of equal growth
r a t e in m e s o c o s m s (d)
5--+-- A
_.co_ B
4-
o
9.A-. C
+
{D
o
+
-4
<
z
o
++
~2
in D W (fix)
S L (fix)
in O T O (fix)
S L (live)
In D N A
Gpi (from
RNA: DNA)
40
40.5
43.5
44
43
19
21
22.5
24
25
26
The polynomial size-at-age relationships within both
mesocosms were time derived to obtain the corresponding growth rates. The rate of increase of larval
length, dry weight and total DNA content was initially
higher in Mesocosm B than in Mesocosm A, but eventually became higher in Mesocosm A than B (Fig. 5a,
b). In both mesocosms the range in average specific dry
weight growth rates between Day 10 and 40 were
higher than for the average specific total DNA growth
rates and the average protein growth rates. In Mesocosm B the growth rates in this period dropped from
13 to below 0% d -~ (Fig. 5a). In Mesocosm A the
average specific growth rate of dry weight increased
from around 2 up to 15% d -~, whereas the average
specific growth rate of total DNA and protein growth
rate increased from around 3 up to 11% d-1 (Fig. 5a).
The age of equal growth rates in the two mesocosms
was estimated as above by the intersection of the
polynomial fits of the respective growth-at-age equa-
z
t-.L~_L.'~ ~+;'* §
§
I
O
+
9
+
O
§
I
10
2;
3;
2o
go
60
Age (days)
F i g . 4. Clupea harengus. A g e - s p e c i f i c R N A : D N A r a t i o s o f h e r r i n g
l a r v a e in M e s o c o s m s A a n d B. Lines r e p r e s e n t p o l y n o m i a l fits
t o t h e d a t a ( T a b l e 2). T h e r e g r e s s i o n e q u a t i o n f o r t h e s t a r v a t i o n c o n t r o l , C , is: R N A : D N A
= 3 . 2 8 8 - 0.551 X - 0 . 0 2 6 X 2,
R z = 0.788, n = 37, w h e r e X is a g e (d)
tions (Fig. 5a, b). The specific growth rate in dry weight
was similar in the two mesocosms around Day 19, and
the total DNA content of the herring larvae was similar
around Day 25 (Fig. 5a; Table 3). The average protein
growth rates were estimated from R N A : D N A ratios
using the relationship from Buckley (1984), and were
higher in Mesocosm B than Mesocosm A until Day 26,
and vice versa thereafter.
The proportion of herring larvae with protein
growth rates below 2% increased from zero to 100% in
the starvation control group after Day 10 (Fig. 6). In
Mesocosm A the proportion of larvae in poor condition also increased from 0% initially to above 70%
between Day 10 and 15 before it dropped to 0% again
after Day 25 (Fig. 6). In Mesocosm B there was an
596
increasing proportion of larvae in poor nutritional condition after Day 25 (Fig. 6).
The herring collected at the termination of the
experiment, between Day 65 and 67, had somewhat
higher RNA : DNA ratios in Mesocosm A (2.3) than in
Mesocosm B (1.9) (t-test, p < 0.02). There was a significant reduction in the ratio after 3d of subsequent
starvation of the herring in the laboratory, 1.2 in Mesocosm A and 1.4 in Mesocosm B, respectively
(p < 0.05, two-way ANOVA). Two to three more days
of starvation did not result in a further significant drop
in RNA: DNA ratios (nominal reduction of 0.1).
Morphometric and biochemical relations
16|&
144
A---In
DNA.--
--
Gpi . . . .
The morphological relationship between SL and In
DW of herring larvae was linear over the size range
studied (Table 4). The same was also the case with SL
and in DNA (Table 4). This simplifies the comparison
of the estimated population growth rates (SL, In DW
and In DNA. Fig. 5a, b) since the underlying size
measures are all linearly related. The length-specific
increase in total DNA was similar in both mesocosms
(ANCOVA, slope parallel p = 0.33, Table 4), whereas
the length-specific increase in dry weight was higher in
Mesocosm B than in Mesocosm A (ANCOVA, slope
parallel p < 0.001, Table 4). The length-specific increase
in dry weight was higher than the corresponding increase in total DNA in both mesocosms (Table 4). The
variability around the In DW versus SL and In DNA
B
~
"2_
x:
6t
/
, , , , - ~ , ~
~
0
0.8
A~
b
S L (fix) - - - - - S L (live) - - 9 ~ In O T O . . . .
'>,,
E
E
B
!9
0.6"
v
0.4.
t-O
.o
o~ 100.
0.2.
o
10
- -+..- A
80
J
co
0
,#
I
I
15
20
I
I
30
25
Age (days)
-.-A-- C
"O
c
/,",
o~ 6 0
.'
' ~ ' ' ~
35
40
Fig. 5 Clupea harengus. Average age-specific growth rates of herring larvae in Mesocosm A (thick lines) and Mesocosm B (thin lines).
(a) Total D N A and dry weight (D W) growth rates were obtained by
using the time derivative of the polynomial fits in Table 2. Estimated protein growth rates (Gpi) were obtained using the polynomial fit to the Gpi data after transformation from R N A : D N A
values (Buckley 1984, Table 2 in present study). (b) Standard length
(SL) growth rates in m m d 1 [from fixated (Moksness et al. 1995)
and live larvae] and otolith radius (OTO) growth rate in % d - 1
were obtained using same procedures as for (a)
/
/
\
/
o
cz 40
.~_
.'
I
\
; t
,, /
20- ,;' /
g
0-
\
\
\
~,-" ~ , ~ - - ~ : r > ~ _
0
1'0
f
_+_ _ ~__ § _ _ _ _.+
a'0
2~0
5;
4'0
60
Age (days)
Fig, 6 Clupea harengus. Percent of herring larvae starving or in
poor condition (defined as larvae with protein growth rates below
2%) in Mesocosms A and B and starvation control, C. Data are
grouped in 5-d intervals
Table 4 Clupea harengus. Morphometric relations in herring larvae in Mesocosms A and B. Numbers in parentheses represent SE of
respective parameters and SE of estimate is the standard deviation around the regression line. ANCOVA results for comparisons between the
respective relations in the two mesocosms are also given
Mesocosm
Regression equations
R2
n
SE
est.
p slope
parallel
A
B
In D W = - 4.552 (0.070) + 0.230 (0.004) SL (fix)
In D W = - 5.067 (0.038) + 0.257 (0.002) SL (fix)
0.963
0.989
158
125
0.214
0.167
< 0.001
A
B
in DNA = - 2.287 (0.118) + 0.211 (0.006) SL (live)
In D N A = - 2.341 (0.073) + 0.203 (0.005) SL (live)
0.891
0.950
136
99
0.183
0.184
0.33
A
B
in RNA = 0.527 (0.024) + 1.388 (0.027) In D N A
in RNA = 0.788 (0.030) + 1.089 (0.021) In D N A
0.945
0.927
161
208
0.294
0.273
< 0.001
p level
< 0.001
597
versus SL regression lines as determined by the SE of
estimate were similar, indicating that total D N A as
analysed with the present methodology is a relatively
precise measure of larval size (Table 4).
The In RNA versus In D N A regressions had different
slopes in the two mesocosms (ANCOVA, p < 0.001,
Table 4). The rate of increase in RNA per unit of D N A
was higher in Mesocosm A than in Mesocosm B.
A common linear regression was fitted to the combined
data sets from the mesocosms. The residuals from this
common regression showed a similar temporal pattern
as the R N A : D N A ratios in the two mesocosms
(Fig. 7). The crossing of the polynomial fits indicate
that the larvae were in similar condition around Day 22.
R N A : D N A ratio as a growth predictor
The R N A : D N A ratio emerged as a significant factor in
all four stepwise regressions describing growth rates of
1.0 84
0.6 84
-+- A
-c-B
F~ Bo
R
R
O
oB=
[]
[]
o+
++
++
+
+
+
--~ 0.2
"(3
~ -0.2
"~~*~g~0~~
.......... = < .... * -
x_
-0.6
u
*+
*$++
D +
up
B
~
+
[]
+
+
-1.0
+
-1.4
+
1'o
,
~ I
20
Gdi -- 2.912 + 0.646 Gpi,
[
3'o
40
larvae in the two mesocosms as a function of temperature, size and RNA:DNA ratio (Table 5). Best fits were
obtained in modelling otolith growth rate and D N A
growth rate, and about 70% of the variance was explained in these two models (Table 5). Temperature
contributed significantly to the model describing D N A
growth, whereas a size term was included in the models
describing length, dry weight, and otolith growth. Temperature and size were generally negatively correlated
(r < - 0.7), and regular regressions where the size term
was exchanged with a temperature term thus gave
relatively similar R2-values to the alternative model
(0.557, 0.339 and 0.674 in the length, dry weight, and
otolith models, respectively). The D N A growth model
was the only case where the R N A : D N A and the estimated size (ln DNA) and population growth rate values
were obtained from the same subsample of larvae, and
a more precise relationship was therefore expected. The
mesocosm specific relation between RNA : DNA, temperature and D N A growth rate was also more precise
than the overall relationship based on the combined
data from the two mesocosms (Table 5), and R 2 equalled 0.904 and 0.734 for the models describing growth in
Mesocosms A and B, respectively. RNA: D N A ratio
was the main explanatory variable in Mesocosm A, and
temperature in Mesocosm B (Table 5).
The regression parameters in the D N A growth
model were used to predict D N A growth in the same
manner as Buckley (1984) estimated protein growth
index (Gpi). The dependent variable was termed Gdi,
the D N A growth index, using the relation from both
mesocosms combined in Table 5. Gdi was linearly
related with Gpi:
60
A g e (days)
Fig. 7 Clupea harengus. Residuals from the c o m m o n regression of
In R N A versus In D N A plotted against age in days of herring larvae
from M e s o c o s m s A and B. C o m m o n regression is given as:
In R N A = 0 . 6 0 0 + 1 . 2 3 1
in D N A , R 2 = 0 . 9 3 6 , n = 3 6 9 , SE
est. = 0.312. Polynomial fits are included to show temporal trend in
the data
with n = 41, R 2 = 0.994, SE est. = 0.144.
The R N A : D N A based growth indices (Gpi, Gdi)
provided growth estimates that closely resembled other
independent growth measures when compared in
a PCA analysis (Fig. 8). The first axis in the PCA
analysis can be interpreted as representing a growth
factor, with correlations between the various growth
Table 5 Clupea harengus. Equations of growth rates, G(SIZE), of herring larvae in both mesocosms based on stepwise regressions with the
estimated growth variable as the dependent variable (from polynomial regressions) and R N A : D N A ratio, temperature and size as
independent variables. Mesocosm-specific growth rates are estimated by ordinary regressions
Regression equations
Both mesocosms
G(SL fix) = 0.137 + 0 . 3 1 5 R N A : D N A - 0.033 SL
G(ln DW) = - 8.757 + 6 . 1 5 7 R N A : D N A -- 2.528 in D W
G(ln OTO) = 4.363 + 3 . 3 5 7 R N A : D N A -- 2.098 In O T O
G(ln D N A ) = - 9.921 + 3 . 0 4 6 R N A : D N A + 0.678 T E M P
Mesocosm A
G(ln DNA) = 1.954 + 3 . 2 1 4 R N A : D N A - 0.136 T E M P
Mesocosm B
G(ln DNA) = - 9.904 + 0 . 0 5 9 R N A : D N A + 1.225 T E M P
R 2
n
SE est.
0.652
0.483
0.731
0.699
28
28
28
28
0.100
2.653
0.981
1.205
0.904
14
0.794
0.734
14
0.924
598
1.00.8
DNA SL
oo
o
OTO ~
AGE
o
RNA:DNA
o~ 0.6
o
0.4
Gpi
0.2
~ i
GDNAo
GOTOo
O
0 84
GDWo GSL
o
-0.2
0
012
0',4
0'.6
0'.8
1'0
Factor 1 (47.7%)
Fig. 8 Clupea harengus. Factor-loading plot of growth, age, and
size variables used in principal component analysis. Gpi and Gdi
represent protein and DNA growth indices, respectively, and the
other growth variables derived from polynomial regressions are
labelled with a G-prefix. Data from both mesocosms are included
(n = 28). Percentages of total variance explained by each factor are
shown on respective axes
measures and the first factor of around 0.9 (Fig. 8).
Both Gpi and Gdi should thus be considered useful
indicators of growth in herring larvae under a wide
range of feeding conditions. The second PCA axis can
be considered as representing a size factor, with age as
a relatively poor measure of size in this experiment,
reflecting the variability of size-at-age of larvae from
the two mesocosms. R N A : D N A ratio and to a lesser
extent Gpi and Gdi were also correlated to the size
factor (correlations of 0.3 to 0.6).
Discussion
survival in these experiments have ranged from 35 to
93% to Day 39 (Oiestad and Moksness 1981; Wespestad and Moksness 1990; Moksness 1992). The density
of prey in Mesocosm B is also of the same magnitude as
the initial densities in some plastic enclosure experiments ( ~ 1.8 m 3) with herring reported by Oiestad and
Moksness (1981). In these experiments survival was
relatively high in the absence of predators, ranging
from 16 to 47% after 39 d. The critical density of prey
for survival of young herring larvae in the mesocosms
therefore seems to be below 1 prey 1-1, which is substantially lower than what previously was considered
to be the case (McGurk 1984).
Survival to the end of the present experiment was
somewhat lower than in other similar experiments; this
can most likely be attributed to poor feeding conditions
during different periods in the mesocosms. Around
27% of the larvae were recovered or sampled in Mesocosm A, which is similar to the estimated proportion
of larvae remaining at Day 15. Subsequent mortality
must therefore have been low, as indicated by the
improving feeding conditions and the low proportion
of larvae in poor nutritional condition during the latter
half of the experiment. In Mesocosm B mortality was
low the first 15 d after release, but increased later on as
the feeding conditions deteriorated. An increasing proportion of the larvae was characterised as starving or in
poor nutritional condition in the latter half of the
experiment in Mesocosm B, but cannibalism among
herring larvae may also have contributed to the mortality, as previously documented by Wespestad and
Moksness (1990). No stomach content analyses were
carried out to confirm if this was the case during our
experiments. The average RNA : DNA ratio of juvenile
herring recovered at the end of the experiment in Mesocosm A was, however, higher compared to those
recovered in Mesocosm B, which is an indication of
superior feeding conditions in Mesocosm A at this
stage.
Environmental conditions and survival
The initial temperature in both mesocosms was above
18~ which is higher than common temperatures in
the habitat of autumn-spawned herring larvae (Blaxter
1985), and the larvae had no visible yolk remains 2 to
3 d after hatching. The initial density of prey in Mesocosm A was very low (below 1 prey 1-1), but similar
densities have been reported during autumn and winter
months in the Georges Bank area (Cohen and Lough
1983). The prey density in Mesocosm B is more characteristic of first feeding conditions of larval herring in the
sea (e.g. Kiorboe and Johansen 1986; Munk et al. 1989;
Fossum and Moksness 1993, 1995), although densities
over 100 nauplii 1-1 have been reported in some larval
feeding areas (McGurk et al. 1993). Naupliar densities
above 10 l-1 have also been observed during previous
enclosure experiments in Mesocosm B with different
spring-spawning herring groups, and estimated larval
Different growth measures
The same temporal patterns of larval growth were
evident in both mesocosms with the various growth
measures employed. Average R N A : D N A ratios
showed the same growth pattern (as Gpi) as the other
somatic growth estimates, and is therefore rightfully
considered a useful independent growth measure of
individual fish larvae (Buckley 1984). Several authors
have cautioned against the application of Buckley's
equation to other species and temperature ranges than
it originally was developed for (Bergeron and Boulhic
1994; Westerman and Holt 1994). Malloy and Targett
(1994) established relations between RNA : DNA ratios
and larval growth in summer flounder, Paralichthys
dentatus, and found RNA:DNA ratios better suited for
growth prediction than five other indices used. Malloy
599
and Targett did not employ Buckley's equation, but
based their relation on a series of calibration experiments to obtain the relationship. This is the recommended procedure, but since no data from such calibration experiments were available for herring larvae, we
used the equation by Buckley (1984) which is derived
from several species, including herring. The actual protein growth values should still be treated with care,
however. McGurk et al. (1992) for example reported
protein growth rates of herring larvae in the field of
over 80% d -1 using Buckley's equation, which is
clearly an overestimate. Future calibration experiments
with herring larvae grown under different temperature
and food regimes are thus strongly recommended.
The strong correlation between the DNA growth
index (Gdi), protein growth index (Gpi) and the other
estimated population growth rates emphasize the utility of the RNA: DNA based growth indices as growth
predictors of field-captured larvae. Gpi and Gdi especially were strongly related; this was due to the marked
correlation of the respective regression parameters in
the growth equations. Since the Gdi relation established in this experiment originated from a different
data set than that used to establish the Gpi relationship
(Buckley 1984), the similarity between the two growth
indices is considered real and not a statistical artifact
caused by both growth models having a temperature
and a RNA : DNA term. The relationship between Gpi
and Gdi indicate that protein growth rate is lower than
DNA growth rate at low growth rates and vice versa at
high growth rates (above 8% d-1). This is in accordance with previous findings in herring and other species
that relatively higher DNA : protein ratios are found in
starved larvae than in fed larvae (Richard et al. 1991;
Mathers et al. 1993, 1994). Due to the strong relationship between Gdi and Gpi, the possibility also exists
that Gpi estimates can be replaced by Gdi estimates
and vice versa, and thus add further value to
R N A : D N A based growth indices of field captured
larvae.
The delayed response in protein or length growth
rate compared to weight growth rate is expected since
weight increase is more closely linked to food intake.
A decrease in dry weight will immediately follow a starvation period, whereas fish larvae will continue to grow
in length under short periods of food deprivation (Ehrlich et al. 1976; Richard et al. 1991). In the case of
protein growth rate or otolith deposition rate, both are
dependent on complicated ongoing metabolic processes, and a response will first be noticeable after
a change in endolymph environment or ribosome number and activity (Secor and Dean 1992; Ferron and
Leggett 1994). The larval populations in the two mesocosms had equal dry weight growth rates 2 d before
they had equal length growth rates, and yet another
1.5 d before they had equal otolith growth rates (fixated
larvae, Moksness et al. 1995). This lag in otolith growth
versus length growth is similar to the lag in DNA
growth versus length growth observed in the present
study. The two larval populations obtained equal DNA
and protein growth rates 1 to 2 d after they obtained
equal length growth rates (live larvae). Recent experiments on herring larvae have shown, however, that
RNA activity increases in less than 2 h after re-feeding
(Houlihan et al. 1995), and thus indicate a shorter delay
in protein growth rates than suggested in our study.
An elevated dry weight growth rate was evident
between Day 20 and 40 compared to the other growth
measures in Mesocosm A. This could to some extent be
due to incidental inclusion of relatively larger larvae
among the larvae used for otolith analysis (Moksness
et al 1995), and this has been confirmed by analysis of
another subsample of fixated larvae (K. Rukan, University of Bergen, personal communication 1996). The
length and the length growth rate of the fixated larvae
were also consistently larger in this time interval compared to the corresponding values for the larvae that
were measured alive and subsequently used in the
RNA : DNA analysis. A closer correspondence between
the growth estimates based on R N A : D N A ratios and
total DNA content is expected since they were obtained
from the same larvae. An improved design of the experiment would have been achieved if all the growth
measures were obtained from the same larvae. The
elevated protein growth rates in Mesocosm B close to
Day 40 compared to the other population growth rates
has to be viewed in light of larval avoidance towards
the sampling gear at this stage (Moksness et al. 1995),
and can reflect a more accurate growth estimate of the
sampled larvae.
The RNA : DNA ratios of the initial starvation control quickly dropped below 1 which is well below the
critical level proposed by Clemmesen (1989). The drop
in R N A : D N A ratios of larvae and juveniles collected
at the termination of the experiment was significant
after 3 d of starvation, which is in line with the 3- to 4-d
response time previously observed in herring (Ubersch~ir and Clemmesen 1992; Clemmesen 1994). Similar
response times have also been found in summer flounder larvae (Bisbal and Bengtson 1995) and the larvae
and juveniles of sole, Solea solea (Richard et al. 1991).
The response time is possibly temperature dependent,
and different R N A : D N A ratios have been observed
between fed and subsequently starved larvae reared at
temperatures around 20~ or higher after 1 to 2 d
[-striped bass, Morone saxatilis (Wright and Martin
1985); red drum, Sciaenops ocellatus (Rooker and Holt
1996)]. The estimated protein growth rate of herring in
the initial starvation control was - 1 to - 2% d-1
which is a less pronounced decrease in somatic tissue
than observed for dry weight in starving herring and
cod larvae (McGurk 1984; Folkvord et al. 1994). Reduction in larval dry weight in these studies was around
5% d-1, and Buckley's equation may thus be unsuitable for herring larvae under severe starvation conditions.
600
Size-specific RNA and DNA effects
The RNA:DNA ratio in this study was significantly
correlated with other growth measures, and to a lesser
degree, although significantly, to larval size. A sizedependent R N A : D N A relation in herring larvae has
been suggested by Clemmesen (1994), who found an
increasing ratio with larval size. Stage-dependent
RNA: DNA ratios have also been reported in sole, and
care should thus be taken when comparing ratios of
fish from different stages (Richard et al. 1991). Different
R N A : D N A ratios have been found in different tissue
types (Bulow 1987; Foster et al. 1993), and an allometric increase of different tissue types can contribute to
a size-dependent trend in RNA: DNA ratios. It is also
possible that size-selective mortality can result in
a higher average RNA: DNA ratio with increasing age.
It is not clear, however, to what extent size-dependent
mortality of weak and small individuals contribute to
this observed trend in Clemmesen's study since no
growth or survival curves are presented (Clemmesen
1994). An increase in R N A : D N A values of field captured herring larvae with increasing larval size was also
reported by McGurk et al. (1992), but prey concentration increased simultaneously making it difficult to
infer inherent size-specific effects in RNA : DNA ratios.
The apparent size effect on RNA:DNA ratios in this
study, can to a certain extent be due to the decreasing
temperature in the mesocosms during the experiment.
According to Buckley (1984), R N A : D N A ratio and
temperature are negatively correlated at constant protein growth rates. Temperature and larval size were
strongly negatively correlated (r < - 0.7) in this study,
and thus it is difficult to separate size effects from
temperature effects. Further work is therefore needed
to clarify the role of larval size on RNA : DNA ratios in
herring larvae.
Several authors have advocated the use of RNA
versus DNA regression residuals instead of
R N A : D N A ratios due to the undesirable statistical
features associated with error multiplication in ratios
and due to size-dependent trends in RNA: DNA ratios
(Suthers 1992). Similar use of residuals from length
versus weight regression have long been used as condition indices in fish larvae (e.g. Koslow et al. 1985), and
the residuals from RNA versus DNA regressions can
also be interpreted as deviations from an average condition. It is important that the regressions applied fit
the data adequately over the entire size range studied
(linear relation after in transformation in our case),
since deviations in the fit will result in inaccurate classification of larval condition.
The morphological relation between standard length
and In dry weight was linear over the size range studied;
this implies that regular condition indices of length
versus weight are strongly size dependent (Westernhagen and Rosenthal 1981; McGurk 1985). The
same applies to the relationship between standard
length and in total DNA content, whereas the in RNA
content was allometrically related to In DNA content.
The length dependent increase in total DNA content in
herring larvae reported in this study is within the range
reported in other studies (Fukuda et al. 1986; Clemmesen-Bockelmann 1992; McGurk et al. 1992;
McGurk and Kusser 1992). A common understanding
of expected length-specific total DNA content is, however, still lacking. Some of the discrepancy between
field-captured and laboratory-reared larvae may be
due to the common observation that laboratory-reared
herring larvae are heavier per length unit than fieldcaptured larvae (McGurk 1985).
Rearing methodology
The majority of other experiments validating the utility
of R N A : D N A ratios as growth indices in fish larvae
have been carried out in smaller scale laboratory experiments (e.g. Buckley 1984; Clemmesen 1989, 1994;
Richard et al. 1991; Mathers et al. 1994; Bisbal and
Bengtson 1995). The larvae from some of these experiments have been characterised by relatively slow
growth in spite of food being offered in excess (Clemmesen 1994; Mathers et al. 1994). The problems seem to
be partially related to an inadequate feeding protocol
involving the use of suboptimal prey organisms such as
Brachionus and Artemia spp. Although the nutritional
value of these organisms can be significantly improved
by nutritional enrichment with current methodology, it
is common that they do not promote high survival and
growth rates during the initial larval phase in many
temperate marine fish species (Stmtrup 1993; Folkvord
and Moksness 1995). Another characteristic of most
laboratory rearing experiments is the relatively high
prey densities required for larval growth and survival
compared to in the field or in larger outdoor mesocosms (Oiestad 1990). In our experiment a significant
proportion of the herring larvae was able to survive at
initial prey densities well below 1 prey 1-1 in Mesocosm
A. Common rearing practice in the laboratory involves
prey densities 100 to 1000 times higher than in our
study (McGurk 1984). Thus the present shortcomings
of the small scale laboratory rearing methodology are
important to keep in mind when interpreting results
from laboratory experiments dealing with suitability of
nutritional indices of marine fish larvae.
Rearing of fish larvae in large outdoor mesocosms
generally provides large numbers of fish larvae growing
at a rate comparable to in the field at realistic prey
densities (Oiestad 1990). This advantage is not without
compromise, however. As in this experiment, the environmental conditions, like temperature, are rather poorly controlled. A general decline in rearing temperature
during the rearing of autumn spawned species is as
common as an increase in rearing temperature during
rearing of spring spawners (e.g. r
and Moksness
601
1981; Folkvord et al. 1994; van der Meeren and Nmss
1994). Although this is a common situation for naturally occurring fish larvae in the field, the temperature
increase/decrease may be amplified in mesocosms compared to in more open systems. The relatively strong
temperature gradients during the experiment make it
virtually impossible to separate temperature effects
from larval size effects due to the strong correlation
between the two variables. In addition, marked thermoclines in large outdoor mesocosms will give a range
of possible rearing temperatures for the larvae. Since
the relation between R N A : D N A ratio and protein
growth rate is temperature dependent (Buckley 1984),
this can add variability to the protein growth rate
estimates. Controlled laboratory experiments with
larvae reared under a constant temperature regime will
usually be the preferred method to investigate sizerelated effects without interfering temperature effects.
Conclusions
The use of RNA : D N A ratios and their derived growth
indices provided useful measures of larval growth in the
two mesocosms. Residual analysis of in RNA and in
D N A regression also revealed the same temporal pattern as the RNA : D N A ratios, but with a shorter temporal lag following the somatic growth measures. The
total D N A content provided a size measure of herring
larvae of comparable precision as other size measures,
and the population increase in total D N A content was
similar to that of other size measures such as standard
length and dry weight. D N A growth is given as:
Gdi = 0.68 T E M P + 3.05 RNA:DNA - 9.92. The use
of outdoor mesocosms in larval research is strongly
promoted due to its resemblance to natural conditions
in terms of larval growth dynamics, and the combination of mesocosm experiments with controlled laboratory experiments are expected to be of importance for
the assessment of larval growth dynamics.
Acknowledgements The constructive comments from C. Booman
and G. Nyhammer and two anonymous referees on a previous
version of the manuscript are appreciated. We are also grateful to
colleagues at the Marine Laboratory, Aberdeen, United Kingdom,
who kindly provided us with fertilised herring eggs, and Prof. A.J.
Raae for helpful advice on the analysis of nucleic acids. Partial
funding for this project was provided by the Norwegian Research
Council.
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