The Journal of Experimental Biology 206, 1337-1351 (2003)
Copyright © 2003 The Company of Biologists Limited
doi: 10.1242/jeb.00262
Freshwater environment affects growth rate and muscle fibre recruitment in seawater stages of Atlantic salmon (Salmo salar L.)
Ian A. Johnston1,*,
Sujatha Manthri1,
Richard Alderson2,
Alistair Smart3,
Patrick Campbell2,
David Nickell4,
Billy Robertson3,
Charles G. M. Paxton5 and
M. Louise Burt5
1 Gatty Marine Laboratory, School of Biology, University of St Andrews, St
Andrews, Fife, Scotland, KY16 8LB, UK
2 BioMar Ltd, North Shore Road, Grangemouth Docks, Grangemouth, Scotland,
FK3 8UL, UK
3 Marine Harvest Scotland Ltd, Craigcrook Castle, Edinburgh, Scotland, EH4
3TU, UK
4 Roche Vitamins Ltd, Heanor, Derbyshire, England, DE75 7SG, UK
5 Research Unit for Wildlife Population Assessment, School of Mathematics
and Statistics, University of St Andrews, St Andrews, Fife, Scotland, KY16
9LZ, UK
*
Author for correspondence (e-mail:
iaj{at}st-andrews.ac.uk)
Accepted 23 January 2003
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Summary
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The influence of freshwater environment on muscle growth in seawater was
investigated in an inbred population of farmed Atlantic salmon (Salmo
salar L.). The offspring from a minimum of 64 families per group were
incubated at either ambient temperature (ambient treatment) or in heated water
(heated treatment). Growth was investigated using a mixed-effect statistical
model with repeated measures, which included terms for treatment effect and
random fish effects for individual growth rate (
) and the instantaneous
growth rate per unit change in temperature (
). Prior to seawater
transfer, fish were heavier in the heated (61.6±1.0 g; N=298)
than in the ambient (34.1±0.4 g; N=206) treatments, reflecting
their greater growth opportunity: 4872 degree-days and 4281 degree-days,
respectively. However, the subsequent growth rate of the heated group was
lower, such that treatments had a similar body mass (3.7-3.9 kg) after
approximately 450 days in seawater. The total cross-sectional area of fast
muscle and the number (FN) and size distribution of the fibres was
determined in a subset of the fish. We tested the hypothesis that freshwater
temperature regime affected the rate of recruitment and hypertrophy of muscle
fibres. There were differences in FN between treatments and a
significant agextreatment interaction but no significant cage effect
(ANOVA). Cessation of fibre recruitment was identified by the absence of
fibres of <10 µm diameter. The maximum fibre number was 22.4% more in
the ambient (9.3x105±2.0x104 than in
the heated (7.6x105±1.5x104)
treatments (N=44 and 40 fish, respectively; P<0.001). For
fish that had completed fibre recruitment, there was a significant correlation
between FN and individual growth rate, explaining 35% of the total
variation. The density of myogenic progenitor cells was quantified using an
antibody to c-met and was approximately 2-fold higher in the ambient than in
the heated group, equivalent to 2-3% of the total muscle nuclei. The number of
myonuclei in isolated fibre segments showed a linear relationship with fibre
diameter. On average, there were 20.6% more myonuclei in 200-µm-diameter
fibres isolated from the ambient (3734 myonuclei cm-1) than from
the heated (3097 myonuclei cm-1) treatments. The maximum fibre
diameter was greater in heated than in ambient groups, whereas the
agextreatment interaction was not significantly different (ANCOVA).
There were also no consistent differences in the rate of hypertrophy of muscle
fibres between treatments. It was concluded that freshwater temperature regime
affected fibre number and the nuclear content of fast muscle in seawater but
not the rate of fibre hypertrophy. The mechanisms and life history
consequences of developmental plasticity in fibre number are discussed.
Key words: muscle growth, myogenic cells, muscle fibre recruitment, temperature, growth, developmental plasticity, fish, Salmo salar
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Introduction
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Many species of teleost fish exhibit indeterminate growth
(Weatherley and Gill, 1987
).
Muscle fibres reach a maximum diameter, which is dictated by metabolic demands
and diffusional constraints related to nutrient assimilation and/or metabolite
removal. An important consequence of indeterminate growth is that the number
of fibres needs to increase throughout much of the life cycle as the muscle
mass increases, involving a prolonged period of postembryonic hyperplasia
(Greer-Walker, 1970;
Stickland, 1983). Species that
reach a large ultimate body size require more muscle fibres than do small
species, and therefore hyperplasia continues for longer in the life cycle
(Weatherley et al., 1988).
Variations in the duration of muscle fibre recruitment also underlie sexual
dimorphism in body size. For example, in female Argentine hake (Merluccius
hubbsi), fibre recruitment ceased at 55-60 cm total length (TL) compared
with 45-52 cm TL in males, reflecting their greater maximum size
(Calvo, 1989). Postembryonic
muscle growth in fish involves a highly proliferative population of myogenic
progenitor cells that have many of the characteristics of the satellite cells
found beneath the basal lamina of muscle fibres in mammals
(Koumans and Akster, 1995;
Fauconneau and Paboeuf, 2001).
However, some myogenic cells are located outside the basal lamina in the
larval and juvenile stages of teleosts
(Veggetti et al., 1990;
Johnston, 1993). The pool of
proliferating myoblasts donates a large number of nuclei to muscle fibres as
they expand in diameter (hypertrophic growth;
Koumans et al., 1991).
Two main processes of fibre recruitment have been identified in teleost
myotomal muscle. In the late embryo, larval and early juvenile stages, muscle
fibres are added from discrete germinal zones
(Veggetti et al., 1990;
Rowlerson et al., 1995;
Johnston et al., 1998;
Barresi et al., 2001) in a
process that has been termed `stratified hyperplasia'
(Rowlerson and Veggetti,
2001). The anatomical location of these germinal zones varies
among muscle fibre types and species
(Rowlerson et al., 1995;
Johnston et al., 1998). In
most fish, the final and most important mechanism of muscle expansion is
`mosaic hyperplasia' (Rowlerson and
Veggetti, 2001). Mosaic hyperplasia involves the widespread
activation of myogenic precursors scattered throughout the myotome
(Koumans and Akster, 1995;
Johnston et al., 1999;
Johnston, 2001). Proliferating
myoblasts withdraw from the cell cycle and fuse to form myotubes on the
surface of existing muscle fibres
(Veggetti et al., 1990;
Johnston et al., 1998,
2000a). In the fast muscle,
immature muscle fibres have a higher content of glycogen and aerobic enzymes
than do mature ones, resulting in a characteristic mosaic pattern of fibre
diameter and histochemical staining characteristics
(Johnston et al., 1975;
Matsuoka and Iwai, 1984;
Rowlerson et al., 1995). Cells
with the ultrastructural characteristics of undifferentiated myoblasts are
first observed after the completion of segmentation and the formation of the
embryonic muscle (Atlantic herring Clupea harengus,
Vieira and Johnston, 1992;
rainbow trout Oncorhynchus mykiss and pearl fish Rutilus frisii
meidingeri, Stoiber and Sänger,
1996).
The number and diameter of muscle fibres present in the myotomes from
hatching to first feeding has been shown to vary with egg incubation
temperature in such diverse species as Atlantic salmon (Salmo salar;
Stickland et al., 1988;
Johnston and McLay, 1997),
whitefish (Coregonus lavaretus;
Hanel et al., 1996), rainbow
trout (Matschak et al., 1998),
Atlantic herring (Vieira and Johnston,
1992), plaice (Pleuronectes platessa;
Brooks and Johnston, 1993),
turbot (Scophthalmus maximus;
Gibson and Johnston, 1995) and
Atlantic cod (Gadus morhua;
Galloway et al., 1998). The
muscle fibres present in these early stages of ontogeny reflect the processes
of embryonic myogenesis (Devoto et al.,
1996; Johnston,
1993; Veggetti et al.,
1990) and fibre recruitment by stratified hyperplasia
(Johnston et al., 1998;
Barresi et al., 2001). The
number of myogenic precursors identified by ultrastructural criteria was found
to be significantly higher in 1-day-old Atlantic herring larvae reared at
8°C than at 5°C (Johnston,
1993). Furthermore, after 80 days at ambient temperature, there
were more fast muscle fibres in herring larvae hatching from eggs incubated at
8°C than at 5°C, consistent with an effect of development temperature
on mosaic hyperplasia (Johnston et al.,
1998,
2001). The number of fast and
slow muscle fibres has also been reported to vary with development temperature
throughout the larval stages of sea bass Dicentrarchus labrax
(Ayala et al., 2001) and in the
freshwater parr stages of Atlantic salmon
(Johnston et al., 2000b). For
salmon, the treatment group with the highest fibre number also had the highest
density of cells expressing c-met (Johnston et al.,
2000a,b),
a molecular marker of myogenic precursor cells
(Cornelison and Wold, 1997).
There is therefore evidence that development temperature has persistent
effects on all three phases of muscle growth.
The recruitment and hypertrophy of fast muscle fibres was shown to vary
with growth rate in rainbow trout fed different ration levels
(Rasmussen and Ostenfeld,
2000). Valente et al.
(1999) found evidence for a
faster rate of fibre recruitment in a fast-growing strain of rainbow trout
compared with a slow-growing strain, although there was a similar number of
muscle fibres at any given body length. Numerous structural and regulatory
genetic loci are likely to influence growth potential in teleosts
(Mommsen and Moon, 2001).
Inter-specific comparisons indicate that a high growth rate and a large body
size are related to the capacity to recruit new muscle fibres
(Weatherley et al., 1988).
Salmo salar L. is a migratory species with a remarkably plastic
and complex life history (Stabell,
1984). Adult stages spawn in freshwater rivers, where the parr
remain for 1-5 years before smolting and migrating to the sea. The salmon then
spend 1-4 years on oceanic feeding grounds in the Arctic before sexually
maturing and migrating back to their natal stream to spawn. In the present
study, we have exploited the availability of an inbred population of farmed
salmon to test two hypotheses concerning postembryonic growth. Our first
hypothesis was that relatively small differences in freshwater temperature
regime have persistent effects on the recruitment and hypertrophy of fast
muscle fibres during growth in seawater. The second hypothesis was that in
fish fed to appetite there would be a correlation between the final number of
muscles recruited and individual growth rate. In order to test these
hypotheses it was necessary to develop a statistical model that accounted for
the effects of scale and temperature on growth rate.
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Materials and methods
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Fish
Atlantic salmon (Salmo salar L.) were from the genetic improvement
programme operated by Marine Harvest (Scotland) Ltd. The hen salmon used were
originally derived from several batches of eggs stripped from wild-caught fish
from the River Shin, Scotland in the late 1960s and early 1970s. The founder
population had been inbred for 9-10 generations. The male fish used were
derived from a single batch of eggs from a late maturing strain of Norwegian
salmon and had been subject to a selective inbreeding programme for seven
generations (since 1973). In order to randomise genetic effects between
treatments, 3-sea winter males were crossed with 2-sea winter females and a
large number of families were generated for the populations studied. 45 hens
and 8 cock fish were stripped to produce 360 families for the `heated' group,
and 32 hens and 2 cocks were stripped to produce 64 families for the `ambient'
group. The eggs were fertilised on 21 November and 30 November 1998 for the
heated and ambient groups, respectively. Following water hardening, the
fertilised eggs were placed in standard concrete hatchery trays at a flow rate
of 10 1 min-1. The water supply to the heated group was maintained
at approximately 8.3°C, which was 1-3°C above that of the ambient
group during the period from fertilisation to pigmentation of the eyes. The
heated and ambient groups hatched 55 days (447 degree-days) and 59 days (457
degree-days) after fertilisation, respectively. The temperature history of the
two treatment groups throughout the entire period in freshwater is shown in
Fig. 1. The heated group were
maintained at warmer temperatures than the ambient group, except for a period
in the summer. The cumulative degree-days in freshwater were 4872 for the
heated group compared with 4281 for the ambient group.
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Fig. 1. The temperature regime experienced by the ambient (open circles) and heated
(filled circles) treatment groups of Atlantic salmon.
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Fish were maintained according to standard hatchery practice and fed a
commercial diet (BioMar Ltd, Grangemouth, Scotland, UK) in duplicate 2 m and
then 4 m circular tanks. The fish were branded when they reached a mass of 10
g and individually PIT tagged (passive integrated transponder tagged; Fish
Eagle Co., Gloucester, UK) prior to seawater transfer to identify groups.
Smolts were stocked into two steel construction sea cages (5 mx5
mx5 m) with a 12 mm mesh cube net at Loch Eil, Scotland, UK on 17/18
April 2000. Each cage was stocked with 600 smolts, representing a random
selection of the available fish from the heated and ambient treatments, which
were age 506 days and 515 days postfertilisation, respectively. Fish were fed
to appetite using an automated system (AKVA smart, UK Ltd, Glasgow, Scotland,
UK) with a standard commercial ration of the Ecolife® series (BioMar Ltd)
manufactured in five pellet sizes (3 mm, 6.5 mm, 9 mm and 12 mm) as the fish
increased in size throughout the experiment. The sea cages were stocked with
Goldsinney wrasse (Ctenolabrus rupestris; 1 per 50 salmon) to control
sea lice infestations. Additional treatments with Excis [1% cypermethrin
(m/v); Novartis Animal Health, Litlington, UK] for 1 h were performed on three
occasions. In all treatments, the net pens were raised to a depth of 1 m and
enclosed in a tarpaulin with oxygen provided to ensure that a minimum level of
7 p.p.m. was maintained.
Sampling strategy
A total of 206 fish from the ambient treatment and 298 fish from the heated
treatment were weighed 11 times in seawater to investigate growth performance
(Table 1). In addition, a set
of fish was randomly sampled for studies of muscle structure on 9 June 1999
(freshwater stage) and 12 July, 3 August and 1 November 2000 and 10 January, 7
March and 4 June 2001 (seawater stages). A non-random selection of the largest
fish in each cage was also made on 4 June and 22 August 2001. Fish were
identified by brand and cross-referenced against PIT-tag number, and their
mass and fork length were recorded.
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Table 1. Numbers of fish by treatment and seawater cage that were repeatedly
weighed to assess growth performance
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Analysis of muscle growth
The fish were lightly anaesthetized in benzocane (20 mg l-1) and
killed with a blow to the head. Muscle blocks were prepared immediately. A 0.7
cm thick steak was prepared at the level of the first dorsal fin ray using a
sharp knife. The entire steak was sampled for fry. For the seawater stages,
the trunk cross-section was traced onto acetate sheets in triplicate using a
fine pen, identifying slow and fast myotomal muscle, the fin muscles and
non-muscle components. The fast myotomal muscle component of the steak from
one side of the body was divided into a series of evenly spaced blocks ranging
from three per individual in the smallest fish to 12 per individual in the
largest fish. Blocks were mounted on cork sheets and frozen in 2-methyl butane
cooled to near its freezing point (-159°C) in liquid nitrogen. The blocks
were wrapped in tin foil and stored in a liquid nitrogen refrigerator until
they could be processed. The blocks were equilibrated to -20°C, and 7
µm transverse frozen sections were cut, mounted on poly-L-lysine-coated
slides, air dried and either stained with Mayer's haematoxylin or used for
immunohistochemistry. Randomly selected fields containing 100-300 muscle
fibres per block were digitised using an image analysis system (SigmaScan
software; SPSS Inc., Chicago, IL, USA), and the mean fibre diameter was
calculated. A minimum of 800 and an average of 1000 muscle fibres were
measured per fish, and the fibre number was estimated from the total
cross-sectional area as described previously
(Johnston et al., 1999). The
maximum fibre diameter in each fish was determined from the mean of the 10
largest measured diameters in the sampled fibres.
Immunocytochemistry
Myogenic cells were identified by their expression of c-met
(Johnston et al., 1999), and
their location was determined using an antibody to laminin
(Koumans et al., 1991), a
major component of the basal lamina of muscle fibres. 18-µm-thick frozen
sections were fixed in acetone for 10 min and then air dried for 10 min.
Non-specific binding sites were blocked in a solution containing 20% (v/v)
normal goat serum, 1.5% (m/v) bovine serum albumin (BSA) and 1% (v/v) Triton
X-100 in phosphate-buffered saline (PBS) (all from Sigma Chemicals, Poole,
UK). Anti-rabbit polyclonal immunoglobulin G (IgG) antibodies to laminin and
m-met were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).
Both antibodies were shown to cross-react with the salmon muscle proteins. The
antibodies were diluted 1:100 (v/v) (laminin) or 1:20 (m-met) in 1% Triton
X-100, 1.5% (m/v) BSA, 10% (v/v) normal goat serum in PBS. The sections were
rinsed in PBS for 5 min and incubated in the primary antibody overnight at
4°C. The sections were washed in PBS and incubated in a 1:800 (v/v)
dilution of the secondary antibody (biotin-conjugated anti-rabbit IgG; Sigma).
The sections were rinsed in PBS and were incubated for 1 h in the dark in a
1:100 (v/v) dilution of extravidin—Cy3 conjugate (Sigma). The sections
were equilibrated in 2xSSC (300 mmol l-1 NaCl, 30 mmol
l-1 sodium citrate, pH 7.0 at 20°C) for 5 min, counterstained
in Sytox green® (Molecular Probes Inc., Leiden, The Netherlands) and then
mounted in a fluorescent medium (DAKO Corp., Carpinteria, CA, USA). The
sections were viewed with a laser confocal microscope (BioRad Radiance 2000).
The density of myonuclei (stained green with Sytox green) and
c-met+ve cells (stained yellow) were quantified in sequential
scanning mode in five or six fields of 0.37 mm2 tissue section per
fish using LaserPix v.4.0 software (BioRad, Hemel Hempstead, UK). Nuclear
counts were corrected for section thickness and the mean diameter of nuclei
(Abercrombie, 1946) previously
determined from electron micrographs
(Johnston et al., 2000a).
Nuclear content of isolated muscle fibres
Small bundles of fast muscle fibres were isolated from the dorsal myotome
just behind the region sampled for histology. Fibre bundles were pinned at
their resting length on strips of Sylgard (RS Ltd, Corby, UK) and fixed for
6-10 h in 4% (m/v) paraformaldehyde in PBS. Single muscle fibres freed from
connective tissue were isolated in PBS using a binocular microscope fitted
with dark-field illumination. Fibres were suspended in 1% (m/v) saponin in PBS
for 3 h, washed three times in PBS and treated with 2 µg ml-1
units RNase (Sigma). Following further washes in PBS, the nuclei were stained
with 30 µmol l-1 Sytox green in PBS for 5 min in the dark.
Fibres were mounted on glass slides using fluorescent mounting medium (DAKO)
and viewed with a laser confocal microscope (BioRad Radiance 2000). The
density of fluorescent myonuclei was quantified in fibre segments 0.3-0.6 mm
long using a z-series of 1 µm optical thick sections and LaserPix v.4.0
software.
The growth model
The specific growth rate (GRt) for a period of time
t is defined as:
 | (1) |
where M0 is the initial body mass and
Mt is the mass after t days.
Mooij et al. (1994)
modelled the expected instantaneous growth rate of a fish
(d
t/dt) as a
temperature-dependent power function of Mt as follows:
 | (2) |
where the parameters are listed in Table
2. For constant temperatures, Elliott
(1975) gave the following
mass—time function (equation 3) as a solution to equation 2:
 | (3) |
where the expected mass after t days,
t, can be calculated given
M0 and the other parameters. After a further
t' days, the expected mass will be given by:
 | (4) |
Mooij et al. (1994) assumed
that ß had the value 0.6 and estimated the parameters and
by minimising the sum of the squared differences between the log-transformed
observed and predicted masses as shown in equation 5, where N is the
number of fish and k is the number of observations for each fish:
 | (5) |
Rearranging equation 4 and using observed, rather than predicted, masses
gives:
 | (6) |
Thus, the change in mass from time t to t' is a
function of time t', temperature and the growth rates
and .
A standard multiple regression equation has the form:
 | (7) |
where y is the response term, x1 and
x2 are the explanatory variables, a is the
intercept, b and c are the regression coefficients and
is the error, or residual, term.
Letting
y=(Mt+t'1-ß-Mt1-ß)/(1-ß),
x1=t' and
x2=t'(T-Tavg) allows
equation 6 to be specified as shown below in model 1:
 | (Model 1) |
Here, we assume that the intercept term is zero so that when time equals zero,
the change in mass will also equal zero. The regression coefficients of model
1 are the growth rates and (defined in
Table 2), and these can be
estimated using multiple linear regression. The units of and
are g1-ß day-1 and g1-ß
day-1 deg.-1, respectively. Models were fitted using
computer programmes written in R, an open source dialect of S-plus.
Model 1 estimates a mean value of the growth rates and , but
a treatment effect can be included as shown in model 2:
 | (Model 2) |
where i=1,...,n and n is the number of treatments.
The change in mass is now a function of time, temperature, growth rates and
treatment. Analysis of variance (ANOVA) was used to determine whether
including a treatment effect significantly improved the fit of the model, thus
indicating whether there was a statistical difference in the growth rate
between the treatments. Similarly, the effects of the duplicate sea cages can
be assessed. In model 3, sea cage was included so that the growth rates
and were now functions of both treatment i and cage
k:
 | (Model 3) |
The parameter of interest is the growth rate and this will vary from
fish to fish. Thus, for an individual fish j, it can be thought of as
being composed of a component due to treatment i, i
from model 2 (or ik from model 3), plus a random component,
Aj. Model 4 incorporates a random effect for fish:
 | (Model 4) |
The random component Aj can then be thought of as a random
variable with a distribution that is assumed to be Normal with a mean of zero
and a variance 
2A [Aj 
N(0,2A)]. Essentially, the term
i is the mean growth rate for treatment i, and
Aj represents the difference between the growth rate of
the jth fish and the mean growth rate for a fish. Thus, the change in
mass is expressed as a function of time, temperature, growth rates, treatment
and fish. Model 4 also takes into account the repeated nature of the
observations; the data consist of several measurements on the same fish and so
each growth period for a particular fish cannot strictly be treated as an
independent piece of information. Including fish as a random component in this
way takes the repeated measurement aspect into account. Since model 4 includes
both random and fixed effects it represents a mixed-effects model.
Model 4 included a random fish effect for but we would also expect
that , the change in instantaneous growth rate per unit change in
temperature, varies randomly for each fish. This is included in model 5:
 | (Model 5) |
where Gj is the random component and is distributed as
N(0,2G).
Statistical analyses
The effect of treatment group on fibre number (FN) and the maximum
fibre diameter in relation to age post-fertilisation was investigated with a
General Linear Model ANOVA with a normal error structure using sequential sums
of squares (MinitabTM statistical software; Minitab Inc., State College,
USA). The model fitted had treatment, age and an age—treatment
interaction term as fixed factors. Cage was nested in treatment, with cage as
a random effect and age post-fertilisation as a covariate. The FN
data were square-root transformed to normalise the residuals. The effect of
treatment and total muscle cross-sectional area (TCA) on FN was
analysed using a similar model with a TCA3—treatment
interaction term, and with TCA as a covariate with cage as a random factor
nested in treatment. The data on the density of nuclei and c-met+ve
cells were analysed using a one-way analysis of covariance (ANCOVA).
Post-hoc testing was by Fisher's least-significant difference test.
Plots of residuals versus fitted values, normal probability
versus residuals and histograms of the residuals were examined for
each of the data sets.
Nonparametric statistical techniques were used to fit smoothed probability
density functions to the measured diameters using a kernel function as
described in Bowman and Azzalini
(1997). The application of
these methods to the analysis of muscle fibre diameters has been described in
detail previously (Johnston et al.,
1999). Values for the smoothing parameter h
(Bowman and Azzalini, 1997)
were in the range of 0.13 to 0.20 with no systematic differences between
groups. Bootstrap techniques were used to distinguish underlying structure in
the distributions from random variation
(Bowman and Azzalini, 1997;
Davison and Hinkley, 1997;
Johnston et al., 1999). The
Kolmogorov—Smirnov two-sample test statistic was used to test the null
hypothesis that the probability density functions of treatment groups were
equal over all diameters. A Wilcoxen nonparametric test was used to test the
hypothesis that the median value of the 50th percentile was equivalent between
groups.
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Results
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Effect of freshwater treatment on seawater growth
The seawater growth of the treatment groups is illustrated in
Fig. 2. Prior to seawater
transfer, fish were heavier in the heated (61.6±1.0 g; N=298)
than in the ambient (34.1±0.4 g; N=206) treatments (mean
± S.E.M.), but their subsequent growth rate was lower. As a result, by
the final sample in July 2001, the masses of the treatments were similar;
3718±75 g for the ambient group (N=176) and 3879±60 g
(N=267) for the heated group. A multiple linear regression approach
was used to model growth rate, where y, the response variable, and
x1 and x2, the explanatory variables,
were defined as shown below in equation 8:
 | (8) |
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Fig. 2. The growth performance of fish from the ambient (open circles;
N=206) and heated (filled circles; N=298) treatment groups
of Atlantic salmon. The values represent means ± S.D. NS, no
significant difference between treatments.
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The analysis of variance for models 1 and 2 is shown in
Table 3. Model 1 estimated a
mean value of the growth rates of all fish, whereas model 2 estimated
different values of the growth rate and the temperature sensitivity of growth
rate for each treatment (Table
4). An F test indicated a significant treatment effect at
the P<0.05 level. The results for model 3 indicate that there was
a significant cage effect (Table
3) but, in practice, the effect of including cage was not large
(Table 4); thus, we chose to
ignore cage effect when fitting model 4 and model 5. The values of the growth
rate in Table 4 show
that the regression coefficient for cage 1 of the heated group was similar to
cage 2 of the ambient group. Model 4 included a random fish effect for growth
rate , and model 5 included a random fish effect for both and
. A log-likelihood ratio test was used to test whether the
more-specific model 5 was required over model 4
(Table 5). Model 5 was
preferred over model 4, and the estimates of the regression coefficients for
model 5 are shown in Table 4.
The fitted values of y for model 5 plotted against
x2 are displayed in
Fig. 3. The green dots indicate
the range in the fitted values because of including a small random value for
each fish. The fit of the model to the observations was generally good but
less so for the first seawater sample (Figs
3,
4). There was a significant
difference between the treatment groups at weighings 1-8 (335 days), but there
was no difference at weighings 9-11. Both instantaneous growth rate ()
and the change in instantaneous growth rate per unit change in temperature
() were significantly higher for the ambient than the heated treatment
groups (Table 4).
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Fig. 3. Fit of the growth model to the observed data of body mass in seawater. In
the figure, y is plotted against x2, where
y=(Mt+t'1-ß-Mt1-ß)/(1-ß)
and x2=t'(T-Tavg). The
variables are defined in Table
2. Black dots represent observed values for the ambient group, red
dots represent observed values for the heated group, and the fitted values of
y from model 5 are shown in green. The values have been offset
slightly along the x-axis so that they can be seen more clearly.
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Fig. 4. Histograms of y for each growth period (i.e. period 1 is between 4
April 2000 and 31 May 2000, period 2 is between 31 May 2000 and 10 July 2000,
etc.; see text for further details).
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Muscle fibre recruitment
The relationship between fibre number (FN) and age
post-fertilisation for a random subset of fish is shown in
Fig. 5A. The final random
sample was taken on 4 June 2001; 904 days and 913 days post-fertilisation for
the ambient and heated groups, respectively. Muscle fibre recruitment was
investigated using ANCOVA (Table
6; model A). The analysis revealed a highly significant difference
in FN between treatment groups (P=0.038, N=129) but
no significant difference between cages. The agextreatment interaction
term was also significant, consistent with a significant effect of treatment
on the rate of muscle fibre recruitment
(Table 6; model A). The
relationship between FN and the total cross-sectional area (TCA) of
fast muscle for the random and non-random (enclosed in box) samples is shown
in Fig. 5B. An ANOVA with
FN as dependent variable, and treatment, TCA and
TCA3xtreatment as fixed effects is also shown in
Table 6 (model B). The
treatment and interaction terms were both significant. A non-random sample of
the largest fish in each cage was also taken in June and on 22 August 2001.
Smooth distributions were fitted to the measurements of fibre diameter in each
sample; an example is shown in Fig.
6. The end of the fibre recruitment phase of growth was identified
by the absence of immature fibres (<10 µm diameter). The maximum number
of fast fibres (FNmax) was calculated by pooling all the
fish that did not contain fibres of <10 µm diameter. The maximum fibre
number was 22.4% greater in the ambient
(9.3x105±2.0x104) than in the heated
(7.6x105±1.5x104) treatments
(N=44 and 40 fish, respectively; F1,82=45.83,
P<0.001; one-way ANOVA). There was a significant correlation
between FNmax and the individual growth rate calculated
from model 4 (June 2001 sample; r2=0.35,
F1,56=29.8, P<0.001;
Fig. 7).
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Fig. 5. The number of fast muscle fibres per trunk cross-section (FN) for
the ambient (open circles) and heated (filled circles) treatment groups of
Atlantic salmon. (A). The relationship between FN and age
post-fertilisation for the randomly sampled fish. The numbers in brackets
represent the number of fish sampled for the ambient and heated groups,
respectively. The arrow shows the age at which smolts were transferred to
seawater cages (SWT). (B) The relationship between FN and the total
cross-sectional area (TCA) of fast muscle at the level of the first dorsal fin
ray. The values represent means ± S.E.M. Fish were selected at random,
except for the last two samples (box), which represents a selection of the
largest individuals available in each cage. The numbers in brackets represent
the number of fish sampled for the ambient and heated groups,
respectively.
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Table 6. Analysis of variance with the number of fast muscle fibres (FN) as a
dependent variable using the method of sequential sums of squares for
tests
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Fig. 6. The distribution of muscle fibre diameter in the fast myotomal muscle of
Atlantic salmon: (A) the final sample of the ambient treatment group sampled
988 days post-fertilisation; (B) the final sample of the heated treatment
group sampled 997 days post-fertilisation. Smooth distributions were fitted to
800 measurements of fibre diameter per fish using a nonparametric kernel
function. The broken lines represent the probability density for individual
fish, and the solid line represents the mean probability density function for
each group.
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Muscle fibre hypertrophy
The first step in the analysis was to investigate the smooth distributions
of fibre diameter. Bootstrap sampling was used to generate a variability band
corresponding to the combined treatments, and this was compared with the mean
probability densities in the heated and ambient groups. The data for the first
and final seawater samples are illustrated in
Fig. 8. In the first seawater
sample (July 2000), the maximum diameter was approximately 165 µm for the
heated group and 145 µm for the ambient group
(Fig. 8A). The right-hand tail
of the distribution, representing the oldest cohorts of fibres recruited in
freshwater, occurred at higher diameters in the heated group than in the
ambient group, reflecting the higher mean body mass of the heated group (225
g) compared with the ambient group (147 g). The maximum density of fibres was
at 25 µm, and this large peak probably represents fibres recruited
following seawater transfer. The peak density in the ambient treatment was
displaced slightly to the left compared with the heated group
(Fig. 8A). Nonparametric
Kolmogorov—Smirnov tests revealed significant differences in the fibre
size distributions between treatments in each of the seawater samples
(P<0.05). In the final sample, illustrated in
Fig. 8B, the mean body mass of
the heated group (4449 g) was not significantly different from that of the
ambient group (4414 g). The right-hand tail of the distribution was at higher
fibre diameters in the heated group than in the ambient group
(Fig. 8B). The peak density of
fibres occurred at approximately 125 µm in the ambient group and 130 µm
in the heated group. The mean probability densities on the left-hand side of
the distribution were very different for heated and ambient groups. The
ambient groups had a higher density of fibres in the range of 50-90 µm
(Fig. 8B), reflecting the
greater intensity of fibre recruitment during seawater growth
(Fig. 5A,B). To investigate the
differences in fibre size further, the 50th percentile (median) fibre diameter
(D50) and the maximum diameter (Dmax)
were calculated. Across the entire experiment, median fibre diameter was
higher for the heated group compared with the ambient group (Wilcoxen test;
P<0.05). The relationship between Dmax and age
post-fertilisation for the randomly sampled fish is illustrated in
Fig. 9A. Fast muscle fibres
approached their maximum diameter of approximately 200 µm at around 800
days post-fertilisation. An ANCOVA with Dmax as the
dependent variable showed a significant effect of treatment but no significant
treatment x age interaction (Table
7). Dmax was higher in heated than in ambient
treatments with respect to body mass (Fig.
9B). There was no consistent difference in the mean rate of
increase in Dmax in seawater between ambient
(0.27±0.06 µm day-1) and heated (0.22±0.06 µm
day-1) treatments. We also used the mean values of fibre number per
group to estimate the number of fibres recruited between successive sample
points. The 800-1000 fibres measured at each sample were ranked by diameter
and then the estimated proportion of fibres recruited since the last sample
was subtracted and the mean diameter of the remaining fibres were calculated.
The estimated rate of fibre hypertrophy of the fibres present at seawater
transfer also showed no consistent differences between treatment groups
(Fig. 10).
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Fig. 8. The mean probability density of muscle fibre diameter for heated (solid
line) and ambient (dashed line) treatment groups for the first (A) and last
(B) seawater samples taken in July 2000 and August 2001, respectively. The
shaded polygon represents 100 bootstrap estimates of the combined populations
of ambient and heated fish, and the dotted line represents the mean
probability density function of the pooled groups. Regions where the mean
probability density function fell outside the shaded polygon provided
graphical evidence for a difference between the populations.
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Fig. 9. The relationship between maximum fibre diameter (Dmax)
and (A) age post-fertilisation and (B) body mass for the heated (filled
circles) and ambient (open circles) treatment groups of Atlantic salmon.
Values represent means ± S.E.M. The number of fish sampled is as in
Fig. 5A.
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Table 7. Analysis of covariance of the maximum diameter of fast muscle fibres in
Atlantic salmon using the method of sequential sums of squares for
tests
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Fig. 10. The rate of muscle fibre hypertrophy of fast muscle fibres between
successive sample points plotted against seawater growth in Atlantic salmon
from the heated (filled circles) and ambient (open circles) treatment groups.
The rate of hypertrophy has been plotted at the midpoint of the time period
over which it was calculated. Values represent the mean of the difference
between the observed fibre diameter and the mean value of the fibre diameter
in the preceding sample.
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Nuclear content of isolated muscle fibres
An isolated single fast-muscle fibre stained for total myonuclei is shown
in Fig. 11A. Connective tissue
was carefully removed from the fibres prior to counting the nuclei. There was
a linear relationship between the number of nuclei in a 1 cm segment of fibre
and fibre diameter (Fig. 11B).
A General Linear Model ANOVA with fibre diameter as covariate and nuclear
content as dependent variable revealed a significant difference between
treatment groups (F1,154=37.48, P<0.001). The
regression equations were as follows: nuclei = 314 + 17.1 fibre diameter
(ambient group; r2=89.3; ANOVA;
F1,75=626.0, P<0.001) and nuclei = 237 + 14.3
fibre diameter (heated group; r2=83.6; ANOVA;
F1,78=398.9, P<0.001). For fast-muscle fibres
of 200 µm diameter, there were 20.6% more nuclei in the ambient treatment
(3734 myonuclei cm-1) than in the heated (3097 myonuclei
cm-1) treatment.
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Fig. 11. The nuclear content of isolated muscle fibres. (A) A fast muscle fibre
stained with the fluorescent DNA stain Sytox green. The confocal image
represents a projection of 1 µm sections through the fibre. (B) The number
of myonuclei in single muscle fibres of 1 cm length in relation to muscle
fibre diameter. Open circles represent the ambient group and filled circles
represent the heated group. The lines represent linear regressions (see text
for details).
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Myogenic progenitor cells
In some cases, myogenic cells were surrounded by the basal lamina of muscle
fibres (yellow arrow in Fig.
12A) whilst in others cases they were not (yellow arrow in
Fig. 12B). The density of
myogenic cells and nuclei was determined for the July 2000 and March 2001
samples (Table 8). ANOVA with
treatment group and body mass as fixed factors and a treatment—body mass
interaction term with body mass as covariate revealed significant differences
between treatment groups with respect to total nuclear density and the density
of c-met+ve cells (Table
8). Differences in nuclear density per mm2 muscle
cross-sectional area reflected differences in fibre size distribution
(Fig. 8) and differences in the
nuclear content of individual muscle fibres
(Fig. 11B). The density of
c-met+ve cells was almost 2-fold higher in the ambient than in the
heated groups, representing 2-3% of the total muscle nuclei
(Table 8).
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Fig. 12. (A,B) Immunohistochemistry showing sections of salmon fast myotomal muscle
double-stained with primary antibodies to laminin and c-met using Cy-3 as the
secondary antibody and Sytox green as a nuclear counterstain. Nuclei are
stained green (white arrowheads), and c-met+ve cells (yellow
arrows) and the basal lamina are stained red.
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Table 8. Density of nuclei and cells immunopositive for c-met in the fast
myotomal muscle of seawater stages of Atlantic salmon reared under `ambient'
and `heated' regimes in freshwater
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Discussion
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For fish fed to appetite, the two most important parameters governing
growth rate are temperature and body size
(Jobling, 1983). Growth
usually shows a distinct optimum related to the life history stage and thermal
tolerance of the species (Weatherley and
Gill, 1987). For example, in Atlantic salmon, the growth rate of
juveniles increased between 6°C and 15.9°C and then declined as
temperature increased to 22.5°C
(Elliott and Hurley, 1997).
The specific growth rate of the fish decreases with increasing body size
(Jobling, 1983). Several
growth models have been developed that provide an estimate of instantaneous
growth rate independent of the effects of temperature and scale
(Ricker, 1979;
Elliott and Hurley, 1997;
Mooij et al., 1994;
Mooij and van Ness, 1998). The
multiple regression model described in the present study incorporated effects
for treatment and cage, and random factors for individual growth rate
() and individual variation in the temperature sensitivity of growth
(). The models provided a good fit to the observed data on fish masses
except for the first period following seawater transfer (Figs
3,
4). It is possible that the
stress associated with the implantation of PIT tags and seawater transfer
depressed growth performance over this first period.
Early thermal experience has been shown to alter the number, size
distribution and nuclear content of muscle fibres in juvenile Atlantic salmon
(Stickland et al., 1988;
Nathanailides et al., 1995;
Johnston and McLay, 1997;
Johnston et al., 2000a,
b). It has been suggested that
such effects are, in part, related to associated changes in oxygen tension due
to the large diameter of salmonid eggs
(Matschak et al., 1998). A
difference in mean egg incubation temperature of 2.8°C was sufficient to
produce changes in muscle cellularity throughout the freshwater parr stages
(Johnston et al., 2000a,
b). In the present study, we
tested the hypothesis that freshwater temperature regime would continue to
have effects on the recruitment and hypertrophy of muscle fibres once the fish
smolted and began a phase of rapid growth in seawater. Salmon were maintained
either in heated water or at ambient temperature until smoltification. The
cumulative degree-days in freshwater were 13.8% higher in the heated groups
than in the ambient groups, providing a significantly greater growth
opportunity. Shortly before seawater transfer, the mean body mass of the
heated groups was, on average, 44.6% more than that of the ambient groups.
Following seawater transfer, the treatment groups were fed to appetite and
reared together in the same cages, providing an equal growth opportunity.
We tested the hypothesis that freshwater treatment would alter the rate of
muscle fibre recruitment and hypertrophy in seawater. In the first seawater
sample, muscle TCA, D50 and Dmax were
all significantly greater in the heated than in the ambient groups, reflecting
the larger body mass of the heated fish. However, the number of muscle fibres
per trunk cross-section was 14.5% higher in the ambient
(3.9x105±1.1x104) than in the heated
groups (3.3x105±6x103)
(P<0.05; Fig. 5A).
An ANOVA revealed significant effects of freshwater environment on the number
of fast muscle fibres throughout the period of seawater growth
(Table 6). The end of the
recruitment phase of growth was identified by the absence of fibres less than
10 µm in diameter. The final fibre number (FNmax) was
22.4% greater in fish reared in ambient compared with heated water
temperatures. These changes in FNmax represent a
freshwater treatment effect on mosaic hyperplasia, since this is the only
mechanism of fibre expansion still active in seawater stages.
Significant differences in the distribution of muscle fibre diameter were
observed between treatments (Fig.
8); however, the reasons for this are complex. The median and mean
fibre diameter were higher in the heated than in the ambient group throughout
the experiment. The median and mean fibre diameters are a reflection of two
opposing processes, namely fibre recruitment and hypertrophy, which tend to
decrease and increase fibre diameter, respectively. It is not possible to
measure the rate of fibre hypertrophy in an individual fish. However, there
were several lines of evidence to suggest that the rate of fibre hypertrophy
in seawater was not affected by freshwater treatment. Firstly, the average
increase in Dmax between samples showed no consistent
trend between treatments. Secondly the age—treatment interaction term
for Dmax was not significantly different
(Table 7). Thus, our first
hypothesis concerning the effect of freshwater environment on seawater growth
was accepted for fibre recruitment but rejected for the rate of hypertrophic
growth.
FNmax is an important anatomical trait because it can
potentially influence both growth rate and maximum body size. The rate of
hypertrophy of individual muscle fibres decreases with age as their maximum
diameter is approached (Weatherley and
Gill, 1987). We found a positive correlation between
FNmax and individual growth rate (), explaining
around 35% of the total variation (Fig.
7). Similar relationships between growth rate and fibre number
within species have been reported previously for mammals (reviewed in
Rehfeldt et al., 1999). In the
present study, the higher fibre number in ambient compared with heated groups
can explain at least some of the difference in growth rate between treatments,
although other causes, including developmental effects on the endocrine system
regulating growth, cannot be excluded.
The mononuclear cells that express the cell surface receptor c-met are
thought to represent a mixture of muscle stem cells and their progeny at
various points along the pathway to terminal differentiation
(Hawke and Garry, 2001;
Zammit and Beauchamp, 2001).
In fish muscle, the majority of c-met+ve cells also express members
of the MyoD gene family, which suggests most are already committed to terminal
differentiation (Johnston et al.,
2000a; Brodeur et al.,
2002). The progeny of the stem cell population either fuse to form
myotubes or are absorbed into existing fibres as they expand in diameter. A
significant fraction of the myogenic cells will also be involved in nuclear
turnover (Schmalbruch and Lewis,
2000). It is not known at what stage the fate of the cells
participating in myotube formation and hypertrophic growth is determined. In
the present study, the number of nuclei in isolated fibre segments was found
to be 20.6% higher in the ambient than in the heated treatments, indicating
that the freshwater temperature regime affected the total production of muscle
nuclei. The number of mononuclear c-met+ve cells representing
myogenic precursors was 2-fold higher in the ambient than in the heated
treatments, equivalent to 2-3% of the muscle nuclei
(Table 8). The proportion of
c-met cells to total nuclei was within the range previously reported for
myosatellite cells identified by electron microscopy in adult common carp
(Cyprinus carpio; Koumans et al.,
1994).
A simple explanation for our results is that the treatment groups differed
in the number of muscle stem cells, resulting in proportional increases in
differentiating myogenic nuclei. However, it is also possible that freshwater
treatment affected some aspect of the signalling pathways that regulate the
proliferation and/or differentiation of myogenic cells. The number of times
the myogenic cells divide prior to terminal differentiation is known to be
influenced by a complex network of hormones, growth factors and transcription
factors, most of which have been poorly characterised in fish
(Johnston et al., 2002).
Growth hormone is a powerful stimulator of insulin-like growth factor-1
(IGF-1) genes, which have multiple actions on myogenic cells
(Florini et al., 1991;
Mommsen and Moon, 2001).
Myostatin (Mstn), a member of the TGF-ß (transforming growth factor
ß) superfamily, is a potent negative regulator of muscle growth
(McPherron et al., 1997) and
is highly conserved across the vertebrates
(Rodgers and Weber, 2001).
Mice carrying a targeted disruption of the Mstn-encoding gene show a 2-fold
increase in muscle mass arising from a combination of increased muscle fibre
hyperplasia and hypertrophy. Mstn-deficient mice also show a suppression of
body fat (McPherron and Lee,
2002), which was not observed in the present study (I. A.
Johnston, unpublished results). The lack of an effect of treatment on fibre
hypertrophy also argues against a role for myostatin and IGF-1 genes
(McPherron et al., 1997;
Barton-Davis et al., 1999).
Previous studies have shown that variations in embryonic temperature regime
alone are sufficient to produce changes in muscle cellularity in fish. There
is evidence that the sensitivity of fibre number to egg incubation temperature
varies between different spawning populations of Atlantic salmon
(Johnston et al., 2000b).
Johnston et al. (2000a)
collected eggs from Atlantic salmon spawning in lowland and upland tributaries
of the River Dee, Scotland, UK. The embryos were incubated at the simulated
temperature regimes of each tributary, which was, on average, 2.8°C cooler
for the upland than the lowland stream. For the lowland fish, FN was
approximately 10% higher when eggs were incubated at the temperature of their
natal stream, whereas in the upland fish FN was similar at both
thermal regimes (Johnston et al.,
2000b). In the present study, eggs from an inbred population of
farmed salmon were incubated at somewhat higher and more constant
temperatures. The choice of constant or fluctuating temperature regime has
been shown to influence the number of muscle fibres in larval pearl fish
(Stoiber et al., 2002).
Without information on the shape of the reaction norm relating fibre number to
development temperature, it is difficult to interpret differences in the
direction of responses among studies. For example, a bell-shaped reaction norm
could produce either an increase, a decrease or no change in fibre number
depending on where on the temperature range the eggs were incubated.
It has been estimated that approximately one-third of the variation in
fibre number in limb muscles of the pig is phenotypic and not related to
genetic origin (Rehfeldt et al.,
1999). Several studies have shown that in mammals poor maternal
nutrition causes a low birth mass and a reduction of the number of secondary
myotubes, resulting in a permanent reduction in the number of muscle fibres
(Wilson et al., 1988;
Dwyer et al., 1995).
Developmental plasticity of muscle growth has been reported previously in
birds prior to the establishment of effective thermoregulation. In this case,
mild heat exposure in young broiler chickens (Gallus domesticus)
resulted in a transient growth halt followed by immediate compensatory growth
(Yahav and Hurwitz, 1996).
Such thermal conditioning was associated with an immediate increase in
circulating IGF-1 concentration followed by satellite cell proliferation
(Halevy et al., 2001).
The temperature prior to hatching is critical for determining the number of
myogenic cells (Johnston et al.,
2000a) and fibre number post-hatch in Atlantic salmon
(Stickland et al., 1988;
Johnston and McLay, 1997;
Johnston et al., 2000b).
Adverse conditions during early development have a negative impact on
subsequent growth in a wide range of species
(Lindström, 1999). The
present study found that FNmax was reduced by 18.3% for a
relatively modest rise in temperature during freshwater development. The
heated treatment could not be considered stressful since growth was increased
relative to the ambient group. Such phenotypic variation in
FNmax in fish for ecologically relevant temperature
changes has the potential to influence a range of life history
characteristics, including growth rate and ultimate body size. However, an
effect of FNmax on ultimate size would require an
independence of fibre number and size. Studies in mammals suggest that this
may not be the case because in animals that have stopped growing, fibre number
and mean diameter are inversely correlated
(Rehfeldt et al., 1999).
Developmental plasticity in growth characteristics may be relatively
commonplace in ectotherms. For example, egg incubation temperature was shown
to influence subsequent growth rate and body size at defined developmental
stages in lizards (Braña and Ji,
2000) and turtles (O'Steen,
1998; Rhen and Lang,
1999), although in these studies the effects on muscle fibre
number were not investigated.
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Acknowledgments
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This work was supported by an industry LINK grant from the Biotechnology
& Biological Sciences (49/LKD12865). We are grateful to Pinney's of
Scotland and AKVAsmart UK for financial support, with special thanks to
Alistair Dingwell and David Whyte for their contributions to project
management. We thank Mr Ron Stuart for expert technical assistance.
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References
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TOP
Summary
Introduction
