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First published online November 19, 2004
Journal of Experimental Biology 207, 4343-4360 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.01292
Rapid evolution of muscle fibre number in post-glacial populations of Arctic charr Salvelinus alpinus
1 Gatty Marine Laboratory, School of Biology, University of St Andrews, St
Andrews, Fife, KY16 8LB, Scotland, UK
2 Holar University College, 551 Skagafjordur, Iceland
* Author for correspondence (e-mail: iaj{at}st-andrews.ac.uk)
Accepted 17 September 2004
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Summary |
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 TOP Summary IntroductionMaterials and methodsResultsDiscussionReferences |
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We tested the null hypothesis that the pattern of muscle fibre recruitment was the same in all morphs, reflecting their recent diversification. The cross-sectional areas of fast and slow muscle fibres were measured at 0.7 FL in 46 DB morphs, 23 LB morphs, 24 PL morphs and 22 PI morphs, and the ages of the charr were estimated using sacculus otoliths. In fish larger than 10 g, the maximum fibre diameter scaled with body mass (Mb)0.18 for both fibre types in all morphs. The number of myonuclei per cm fibre length increased with fibre diameter, but was similar between morphs. On average, at 60 µm diameter, there were 2264 nuclei cm–1 in slow fibres and 1126 nuclei cm–1 in fast fibres. The absence of fibres of diameter 4–10 µm was used to determine the FL at which muscle fibre recruitment stopped. Slow fibre number increased with body length in all morphs, scaling with Mb0.45. In contrast, the recruitment of fast muscle fibres continued until a clearly identifiable FL, corresponding to 18–19 cm in the dwarf morph, 24–26 cm in the pelagic morph, 32–33 cm in the large benthic morph and 34–35 cm in the piscivorous morph. The maximum fast fibre number (FNmax) in the dwarf morph (6.97x104) was 56.5% of that found in the LB and PI morphs combined (1.23x105) (P<0.001). Muscle fibre recruitment continued until a threshold body size and occurred at a range of ages, starting at 4+ years in the DB morph and 7+ years in the LB and PI morphs. Our null hypothesis was therefore rejected for fast muscle and it was concluded that the dwarf condition was associated with a reduction in fibre number.
We then investigated whether variations in development temperature
associated with different spawning sites and periods were responsible for the
observed differences in muscle cellularity between morphs. Embryos from the
DB, LB and PL morphs were incubated at temperature regimes simulating cold
subterranean spring-fed sites (2.2–3.2°C) and the general lakebed
(4–7°C). Myogenic progenitor cells (MPCs) were identified using
specific antibodies to Paired box protein 7 (Pax 7), Forkhead box protein
K1-
(FoxK1-), MyoD and Myf-5. The progeny showed no evidence of
developmental plasticity in the numbers of either MPCs or muscle fibres.
Juveniles and adult stages of the DB and LB morphs coexist and have a similar
diet. We therefore conclude that the reduction in FNmax in
the dwarf morph probably has a genetic basis and that gene networks regulating
myotube production are under high selection pressure. To explain these
findings we propose that there is an optimal fibre size, and hence number,
which varies with maximum body size and reflects a trade-off between
diffusional constraints on fibre diameter and the energy costs of maintaining
ionic gradients. The predictions of the optimal fibre size hypothesis and its
consequences for the adaptive evolution of muscle architecture in fishes are
briefly discussed.
Key words: muscle fibres, myogenesis, growth, myogenic progenitor cell, resource polymorphism, developmental plasticity, fish, Arctic charr, myogenic regulatory factor, Paired box protein 7, Forkhead box protein K1-
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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;
Skúlason et al., 1999).
Glaciers covered most of the current geographical range of the species until
the end of the Pleistocene, around 10 000 years ago. Mitochondrial DNA
sequence variation points to a common Atlantic lineage that diverged from
other charr lineages in the early to mid-Pleistocene, and subsequently
underwent rapid diversification as new freshwater habitats became available at
the end of the last Ice Age (Brunner et
al., 2001). Consistent with this hypothesis, the genotyping of
populations at six microsatellite loci has revealed low levels of genetic
structuring of populations between regions, but high levels between and within
lakes (Wilson et al., 2004).
Wilson et al. (2004) found
that in lakes containing discrete morphs there was evidence for sympatric
populations in ten lakes, and multilocus heterozygote deficits in a further 23
lakes in Iceland, Scandinavia and Scotland, which suggests that separate
breeding populations might be relatively common.
Thingvallavatn (64°11'N, 21°08'W) is situated along an
exposed part of the Mid-Atlantic Ridge system and is Iceland's largest lake
(83 km2). Since its formation approximately 10 000 years ago
tectonic drift and associated volcanic activity has determined its size and
topology, and it is thought to have become isolated from other freshwater
systems approximately 9000 years ago
(Saemundsson, 1992). The
present day lake is dominated by Arctic charr, which are found in most
available habitats. Threespine sticklebacks Gasterosteus aculeatus L.
are also common in the lake, both in sheltered locations and in the
Nitella opaca zone on the bottom. There is also a small population of
brown trout Salmo trutta L. in the lake
(Sandlund et al., 1992). The
four morphs of Arctic charr found in Thingvallavatn
(Fig. 1) represent an extreme
case of phenotypic diversification that is thought to have evolved because of
relaxed interspecific competition for available resources, but increased
intraspecific competiton (Skúlason
and Smith, 1995). Two morphs, a dwarf benthic (DB) and a large
benthic (LB) form, have trophic specialisations for bottom feeding, and can be
identified by having blunt snouts, a sub-terminal mouth and large pectoral
fins (Fig. 1). The DB morph can
effectively exploit the interstitial habitat of lava fissures at the bottom of
the lake for food and shelter whereas LB charr with its larger body size must
forage above the stone matrix (Sandlund et
al., 1992). There are also two `pelagic morphotypes'
(Fig. 1), the planktivorous
(PL) and piscivorous (PI) morphs, which have terminal mouths (longer lower
jaws) and smaller pectoral fins than the benthic morphs
(Sandlund et al., 1992;
Snorrason et al., 1994). The
pelagic morphs fit within the range of morphological variation shown for
Arctic charr in general, and are probably closest to the ancestral condition
(Snorrason and Skúlason,
2004). The four morphs also differ in colouration, growth rate,
foraging behaviour, age at first sexual maturity and maximum body length
(Sandlund et al., 1992).
Female DB morphs start to spawn at 7.5 cm, at 2–4 years of age, and can
reach 27 cm fork length (FL), whereas the female LB morphs start to
spawn at 30 cm, at age 6–10 years, and can reach 54 cm FL
(Sandlund et al., 1992;
Snorrason and Skúlason,
2004). Differences in spawning time, spawning location and
assortative mating behaviours may result in reproductive isolation of the
morphs (Skúlason et al.,
1989a), a hypothesis that is at least partially supported by
genetic data (Wilson et al.,
2004). Local origin of morphs is supported by several studies on
other lakes containing polymorphic charr
(Gíslason et al., 1999;
Wilson et al., 2004). Common
garden rearing experiments indicate that the differences in morphology, growth
and behaviour between the morphs have a strong genetic component
(Skúlason et al.,
1989b,
1993,
1996;
Eiríksson et al.,
1999).
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Fish myotomes contain two or more muscle fibre types arranged in discrete
layers, each with different metabolic and contractile profiles and roles in
swimming (Johnston et al.,
1977). Myotubes are formed in three discrete phases during
ontogeny (Rowlerson and Veggetti,
2001; Johnston and Hall,
2004). Following a purely embryonic phase of myogenesis
(Devoto et al., 1996;
Blagden et al., 1997;
Johnston et al., 1997),
additional fibres are produced from discrete germinal zones by stratified
hyperplasia until the larval or early juvenile stages
(Rowlerson et al., 1995;
Johnston and McLay, 1997;
Johnston et al., 1998;
Barresi et al., 2001;
Johnston and Hall, 2004). The
final and most prolonged phase of myotube formation, termed mosaic
hyperplasia, can continue past sexual maturity and involves a general
activation of myogenic progenitor cells (MPCs) throughout the myotome
(Rowlerson et al., 1995;
Johnston et al., 1998,
2003d).
Body size is expected to have a major impact on the maximum number and size
of the myotomal muscle fibres (Weatherley et al.,
1980,
1988;
Johnston et al., 2003a). In
ten species of North American freshwater fish representing diverse taxa,
muscle fibre recruitment was found to continue until 44% of the maximum fork
length (Weatherley et al.,
1988). Similarly, in species showing marked sexual dimorphism in
body size, such as the Argentine hake Merluccius hubbsi, muscle fibre
recruitment continued for longer in females, which are larger
(Calvo, 1989). In a study of
16 species of notothenioid fish (Perciformes) with sub-Antarctic and Antarctic
distributions, body length was found to explain 69% of the total variation in
the maximum number of fast muscle fibres (FNmax)
(Johnston et al., 2003a).
Antarctic notothenioids contain `giant muscle fibres' reflecting the
relaxation of diffusional constraints at low temperature (Smialovska and
Kilarski, 1981; Egginton et al.,
2002; Johnston,
2003). Phylogenetically based statistical methods revealed a
corresponding and dramatic reduction in size-corrected
FNmax in the lineage leading to the Channichthyidae
(Johnston et al., 2003a).
FNmax in the icefish Chaenocephalus aceratus was
only 7.7% of that in Eleginops maclovinus, a basal sub-Antarctic
notothenioid that reaches the same maximum length
(Johnston et al., 2003a).
Molecular phylogenies provide strong evidence that species from the core
radiation of Antarctic notothenioids have invaded warmer sub-Antarctic waters
over the last few million years (Bargelloni et al.,
1994,
2000;
Stankovic et al., 2001).
However, these species have apparently retained the low fibre number and large
maximum fibre size characteristic of the Antarctic species
(Johnston et al., 2003a).
Since the four morphs of Arctic charr in Thingvallavatn originated in the
lake during the last 10 000 years they provide an excellent model to study
evolutionary processes leading to population formation and possibly speciation
(Skúlason et al.,
1999). Given the great variation in size and growth patterns among
morphs it is interesting to examine possible differences in the evolution of
muscle architecture. In the present study, we tested the null hypothesis that
muscle cellularity was the same in all morphs of Arctic charr in
Thingvallavatn, reflecting their recent diversification. The number of muscle
fibres formed at each phase of myogenesis is sensitive to egg incubation
temperature (Stickland et al.,
1988; Johnston et al.,
2000a,b,
2003c;
Johnston and Hall, 2004).
Variation in spawning time and the influence of cold groundwater on spawning
sites give rise to the possibility of differences in development temperature
between morphs (Skúlason et al.,
1989a). We therefore also investigated the possibility of
temperature-induced plasticity in muscle development between the morphs.
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Materials and methods |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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). The
DB and LB morphs were feeding on the snail Lymnaca peregra and other
benthic invertebrates. The stomachs of the PL and PI morphs contained pelagic
crustaceans and sticklebacks, respectively.
Age determination
The age of the charr were estimated from sacculus otoliths. The otoliths
were read whole in glycerol under a blue light as described by Jonsson
(1976).
Rearing experiments
Eggs from ovulating females of DB, LB and PL morphs were stripped at the
Holar University Aquarium. Eggs from 21 LB, 20 DB and 23 PL morphs were
fertilised in vitro using sperm from a unique male of the same morph.
After 1 h of hydration in water, eggs were split into two and each half
transferred to two development chambers containing incubation troughs
partitioned with 1 mm mesh. Fertilised eggs were separated into families
between partitions. The chambers were fed by a continuous flow of water from
an underground spring and maintained at either 2.2–3.2°C, simulating
the groundwater-fed spawning sites in Thingvallavatn, or in heated water at
4–7°C (range, decreasing during development), simulating the general
lake temperature. The progeny of all morphs experienced identical
temperatures. Eggs were incubated in the dark, and regularly bathed with
Malachite–Green Oxalate (Merk, kGaA; VWR International, Lutterworth,
Leicester, UK) to prevent fungal infection. After hatching, fry were
transferred to 1 m-diameter flow-through tanks, and families were separated
into 0.5 l open containers with a 1 mm-mesh bottom at ambient temperature
(4–6°C) and ambient photoperiod.
Histology
A 0.5 cm transverse steak of the trunk was prepared at 0.7 fork length
(FL) using a sharp knife. The steak was photographed on a light box
using a digital camera and the total cross-sectional area (TCA) of each fibre
type muscle was digitised. Up to 8 blocks (5x5 mm2) were made
from the steak so as to representatively sample all areas of the myotome. The
number of blocks was adjusted to sample 50–100% of one half of the fast
muscle. The blocks included 80–100% of the total cross-sectional of slow
muscle. Blocks were frozen in isopentane (2-methyl butane) cooled to near its
freezing point in liquid nitrogen (–159°C). Frozen sections were cut
on a cryostat at 8 µm thickness.
Enzyme histochemistry
Sections were stained for succinic dehydrogenase activity
(Nachlas et al., 1957) and for
glycogen, using the Periodic Acid Schiff's method
(Pearse, 1960). Sections were
stained for myosin ATPase (mATPase) with and without preincubation at pH 4.3
(30 s to 2 min) or pH 10.2 (30 s to 2 min)
(Johnston et al., 1974).
Electron microscopy
Small bundles of slow and fast muscle fibres were pinned at their resting
length in situ on strips of Sylgard (RS Ltd., Corby,
Northamptonshire, UK) and fixed overnight in 2.5% (v/v) gluteraldehyde, 2.5%
(m/v) paraformaldehyde in 100 mmol l–1 phosphate buffer at
4°C, pH 7.4. Samples were processed for electron microscopy as previously
described (Johnston et al.,
1995).
Antibody preparation
Paired box protein 7 (pax 7) is a transcription factor that can be used as
a marker of myogenic progenitor cells
(Seale et al., 2000). A
full-length pax 7 cDNA was isolated from the fast muscle of the LB
morph by RT-PCR and 5'RACE, cloned and sequenced as previously described
(GenBank; Accession numbers AJ634763-AJ634775; D. Sibthorpe, R.
Sturlaugsdóttir, B. K. Kristjansson, H. Thorarensen, S. Skúlason
and I. A. Johnston, manuscript submitted). Zebrafish pax 7 exonic
sequences were aligned with the Arctic charr pax 7 sequence to
delineate exon/intron borders. Pattern searching of protein sequences was
performed using the PROSITE database. A 13-amino-acid peptide was identified
in the C-terminal region that was conserved among fish and with mouse and was
specific to the Pax 7 protein. The peptide,
H-Gly-Asp-His-Ser-Ala-Val-Leu-Gly-Leu-Leu-Gln-Val-Glu-NH2, was
commercially synthesised, conjugated to keyhole limpet haemocyanin, and used
to immunise two rabbits to provide antisera (Cambridge Research Biochemicals
Ltd, Cleveland, UK).
Other antibodies
Polyclonal antibodies were prepared against peptide antigens designed from
the Atlantic salmon Salmo salar L. MyoD1 sequence (S.
Gottenspare, T. Hansen and I. A. Johnston, unpublished), the Tiger pufferfish
Takifugu rubripes, Forkhead Protein K1- (FoxK1-)
previously called Myocyte Nuclear Factor-) sequence (J. O. Fernandes,
and I. A. Johnston, unpublished) and the common carp Cyprinus carpio
L. Myf-5 sequence (C. M. Martin and I. A. Johnston, unpublished).
FoxK1- is a winged-helix transcription factor that is expressed in
activated MPCs (Bassel-Duby et al.,
1994), whereas MyoD and Myf-5 are members of the MyoD family of
Myogenic Regulatory Factors involved in muscle lineage specification
(Rudnicki and Jaenisch, 1995).
S58 was obtained from the Developmental Studies Hybridoma Bank, University of
Iowa. S-58 is a mouse IgA monoclonal antibody against chicken slow muscle
myosin (Crow and Stockdale, 1968) that cross-reacts with slow muscle myosin in
several teleost species (Devoto et al.,
1996; Johnston et al.,
2003a).
Immunohistochemistry
Frozen sections were fixed in acetone for 10 min and air dried for 10 min.
Myogenic progenitor cells (MPCs) were identified using primary antibodies to
Pax 7, Foxk1-, Myf-5 and MyoD. Slow muscle fibres were identified using
the S-58 antibody. Serial sections were used for single antibody staining.
Primary antibodies were diluted in PST: 1% (v/v) Triton X-100, 1.5% (m/v)
BSA (bovine serum albumin) in PBS (phosphate-buffered saline) as follows: S58
1:10 (v/v), Pax-7 1:2000 (v/v), MNF- 1:1000 (v/v), MyoD and Myf-5 1:800
(v/v). Anti-mouse IgA–biotin conjugate (Sigma, Poole, UK) and
anti-rabbit IgG–biotin conjugate (Sigma) secondary antibodies were
diluted 1:20 (v/v) and 1:800 (v/v) in PST. PST was also used as a dilutent for
blocking and the ExtrAvidin-Peroxidase (Sigma) step. PBS (Sigma) was used for
all washes. Sections were blocked in 5% (v/v) normal goat serum (Sigma) for 1
h, washed in PBS for 5 min and incubated overnight at 4°C in the primary
antibody. After 3x for 3 min washes the sections were then incubated in
the appropriate secondary antibody for 1 h at room temperature, washed again
for 3x 3min and incubated in 1:20 (v/v) ExtrAvidin-Peroxidase for 30
min. The sections were washed 3x for 3 min in PBS and developed using
3-amino-9-ethylcarbazole (Sigma), which produces an insoluble red end product.
The reaction was terminated by washing with distilled water and the slides
were mounted using glycerol gelatine (Sigma).
Determination of the density of MPCs and myonuclei in tissue sections
The density of myonuclei and mononuclear cells stained with Pax 7,
FoxK1-, MyoD and Myf-5 was determined at a magnification of 40x
in 25–40 fields of 0.1 mm2 in each of two blocks where
available (for small fish one block contained the entire trunk cross-section).
The average diameter of 12 nuclei from mononuclear cells was determined from
transmission electron micrographs at a magnification of 19 000 times. Nuclear
counts were corrected for section thickness and the mean diameter of nuclei
(Abercrombie, 1946).
Nuclear content of isolated muscle fibres
Small bundles of fast muscle fibres were isolated from the dorsal myotome
immediately behind the region sampled for histology. Fibre bundles were pinned
at their resting length on strips of Sylgard (RS Ltd.) and fixed for
6–10 h in 4% (m/v) paraformaldehyde, PBS. Single muscle fibres freed
from connective tissue were isolated in PBS solution 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 RNAase (Sigma Chemical, Poole, Dorset).
Following further washes in PBS the nuclei were stained with 30 µmol
l–1 l–1 SYTOX green® (Molecular Probes
Inc, Leiden, The Netherlands) in PBS for 5 min in the dark. Fibres were
mounted on glass slides using fluorescent mounting medium (DAKO Corp.,
Carpinteria, CA, USA) and viewed using a laser confocal microscope (BioRad
Radiance 2000; Hemel Hempstead, Hertfordshire, UK). The number of fluorescent
myonuclei was quantified in fibre segments of 0.3–0.6 mm using a
z-series of 1 µm optical thick sections and LaserPix vs
4.0 software (BioRad, Hemel Hempstead, UK).
Muscle cellularity
S-58 stained sections counterstained with Haematoxyolin were used to
determine muscle cellularity. The cross-sectional areas of a minimum of 600
slow and 1000 fast muscle fibres were measured per fish, sampled equally
between the blocks and the equivalent fibre diameter computed. The total
number of muscle fibres per trunk cross section was estimated as previously
described (Johnston et al.,
1999). Nonparametric statistical techniques were used to fit
smoothed probability density functions (pdfs) 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 0.055 to 0.103 with no systematic variation between morphs.
Muscle fibre recruitment was estimated to have stopped when no fibres in the
smallest size class, 4–10 µm diameter were present. The final number
of fast muscle fibres (FNmax) was the mean ±
S.E.M. of the fibre number estimate for all the fish in which fibre
recruitment had stopped. The 97th percentile of fibre diameter,
calculated from the smooth distributions, was used as an estimate of the
maximum fibre diameter (Dmax).
Statistical analysis
The data were tested for equal variance and normality. Values for
Dmax and FNmax were analysed using a
GML-ANOVA (general linear models-analysis of variance) with morph as a fixed
factor and with either the total cross-sectional area of muscle, fork length
or body mass as a covariate (MinitabTM statistical software; Minitab Inc.,
State College, USA).
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Results |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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).
Muscle fibre types
The composition of myotomal muscle fibres in early life stages was
investigated in the progeny of DB, LB and PL morphs reared in the laboratory.
At hatching, the myotome largely consisted of fast fibres that were unstained
by the S58 antibody (Fig. 2A), and stained weakly for glycogen and succinic dehydrogenase (SDHase) activity
(not illustrated). There was a superficial, 1–2 fibre thick, layer of
muscle that was highly stained for SDHase and glycogen, which extended from
the midline along the whole lateral surface of the myotome (not illustrated).
The superficial fibres at the mid-line stained intensely with the S58 antibody
(s-s in Fig. 2A)whereas those
in the dorsal and ventral regions of the myotome (s-u in
Fig. 2A) were weakly stained or
unstained. Following first feeding, and through the juvenile stages, all the
superficial fibres stained with S58 except for a small number beneath the skin
close to the major horizontal septum. This is illustrated for a 4.5 cm DB
morph in Fig. 2B, in which the
fibres unstained with S58 are labelled with arrowheads. In fish greater than
7.0 cm FL, all the superficial muscle fibres stained with S58
(Fig. 2C). The
S58+ve fibres stained much more intensely for glycogen
(Fig. 2D) and SDHase
(Fig. 2E) than the unstained
fibres. Myosin ATPase staining following 1 minpreincubation at pH 4.3
(Fig. 2F) or pH 10.4 (not
illustrated) revealed two fibre types. Fibres lightly stained for myosin
ATPase stained intensely for S58 and corresponded to slow fibres whereas
fibres unstained by S58 were darkly stained for myosin ATPase, and
corresponded to fast fibres (Johnston et
al., 1975). Although the superficial fibres had a slightly higher
level of staining for glycogen (Fig.
2D) and SDHase (Fig.
2E) than the deeper fibres they were not differentiated on the
basis of either S58 or myosin ATPase activity. Following 90–120 s
preincubation at pH 4.3 the fast muscle showed a mosaic pattern of staining
for myosin ATPase, comprising large darkly staining and small lightly staining
fibres (Fig. 2G). This probably
represents growth stages of fast fibres rather than the presence of distinct
fibre types, reflecting the different composition of fast myosin heavy chain
isoforms in large and small diameter fibres documented previously
(Ennion et al., 1999). Arctic
charr probably have just two main fibre types, based on contractile protein
properties. There were no obvious differences in the composition or relative
amounts of different muscle fibre types in the DB, LB and PL morphs between
hatching and the early juvenile stage.
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Muscle fibre recruitment
The production or recruitment of slow muscle fibres was continuous with
increasing fork length, and fibre number scaled with body mass
Mb0.45, with no significant difference between
morphs (Fig. 3). Slow muscle
fibres with diameters in the range 4–10 µm were surprisingly rare
given the steady increase in slow fibre number with increasing body
length/mass. In juveniles (<10 cm FL), a layer of newly recruited
slow fibres was sometimes observed adjacent to the fast muscle layer at the
major horizontal septum (arrowheads in Fig.
4A), consistent with a discrete germinal zone as described
previously for stratified hyperplasia
(Rowlerson and Veggetti,
2001). However, isolated small diameter fibres could be observed
in all regions of the slow muscle and in all stages examined, including the
largest piscivorous morph examined (Fig.
4B–D). It was concluded that recruitment of slow fibres
continued throughout growth.
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The main method of fibre expansion in the fast muscle was mosaic
hyperplasia. In mosaic hyperplasia, myotubes form on the surface of existing
muscle fibres and mature into small muscle fibres in the size class 4–10
µm. To investigate muscle fibre recruitment in the different morphs, smooth
distributions were fitted to measurements of 1000 fibres per individual using
a nonparametric Kernel function (illustrated for the DB morph in
Fig. 5A; the insert shows an
expanded view of the left-hand side of the distribution). The broken lines
represent the probability density functions (pdfs) of fibre diameter for
individual fish. A total of eight fish, all >18.5 cm FL, had no
fibres in the size class 4–10 µm and were considered to have stopped
myotube production (red lines in Fig.
5A). The median fibre diameter increased, and the right-hand tail
of the distribution progressively moved to the right as fork length increased.
The blue line represents the probability density function of fibre diameter in
the largest DB morph of 27.1 cm FL. Examination of the pdfs of fibre
diameter of the other morphs in relation to fork length (not shown) revealed
that the recruitment of fast muscle fibres ceased at a clearly defined body
length, which was characteristic of each morph. The results of this analysis
are summarised in Fig. 5B,
which shows that muscle fibre recruitment was complete at 18–19 cm
FL in the dwarf benthic morph, 33-34 cm in the large benthic morph,
24–26 cm in the planktivorous morph, and 34–35 cm in the
piscivorous morph. The fast muscle in a 25.7 cm FL DB morph, which
stopped recruiting fibres at 
18 cm, is illustrated in
Fig. 6A. Note the absence of
fibres less than 30 µm. In contrast, a PI morph at 35.8 cm FL
still contained fibres in the size range 10–14 µm reflecting the
longer duration of fibre recruitment with respect to length in this morph
(Fig. 6B). Thus at a given
FL the distribution of fibre diameters differed between morphs
reflecting the different durations of fibre recruitment with respect to
length. The largest DB morph of 27.1 cm had no fast fibres smaller than 45
µm diameter, a situation that was not reached in the PI morph until 50.1 cm
FL (not illustrated).
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The relationship between the number of fast muscle fibres and body length for each morph is shown in Fig. 7. For presentational purposes the data were organised into bins of increasing body size and average values and multidirectional error bars were calculated. An ANOVA with morph as a fixed factor revealed significant differences in fibre number with either FL (F3,110=9.62; P<0.001) or total muscle cross-sectional area (F3,110=17.64; P<0.001) as covariates. The maximum fibre number (FNmax) in each of the morphs is shown in Table 1. Since there was no significant difference between FNmax for the LB and PI morphs the data were combined. FNmax in the DB morph was 56.5% of the value found in the LB and PI morphs combined (P<0.001; one-way ANOVA; Table 1). The data on the largest planktivorous morphs was limited, with the two largest fish of 24 and 26 cm FL having stopped recruiting fibres, consistent with a difference between the PL and PI/LB morphs (Table 1). For individuals showing active muscle fibre recruitment, fibre number scaled with Mb0.51 in the DB and PL morphs combined, and Mb0.31 in the LB and PI morphs combined (P<0.01; Fig. 8).
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A 3-D plot of the relationship between the number of fast muscle fibres in the DB, LB and PI morphs, fork length and age is shown in Fig. 9. The individual fish that have stopped recruiting fast muscle fibres are identified by coloured symbols. The youngest fish that had ended recruitment was a 4+ year class individual in the case of the DB morph, and 7+ year class individuals in the case of the LB and PI morphs (Fig. 9). The cessation of fibre recruitment coincided with a threshold body length and occurred over a range of ages (Fig. 9). Thus, a 9+ year class DB-morph, a 12+ year class LB-morph and a 14+ year class PI morph were caught that were below the threshold fork length and these individuals were still producing fast muscle fibres.
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Muscle fibre hypertrophy
The maximum diameter (Dmax) of each fibre type was
estimated by calculating the 97th percentile of fibre diameter for
each fish from the smooth distributions of fibre diameter. For fish greater
than Mb=10 g, a linear relationship between
log10Dmax and
log10Mb was observed for both fibre types
(Fig. 10). The mass exponent
was 0.18, and no significant difference was observed between morphs. Fish
weighing less than 10 g had a maximum fibre diameter considerable greater than
predicted by these scaling relationships (not illustrated).
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Fibre myonuclei content
The distribution of nuclei in representative isolated single slow and fast
muscle fibre segments is illustrated in
Fig. 11A,B. The majority of
nuclei in the confocal images were found in planes of focus corresponding to
the sub-sarcolemmal zone. The myonuclei content of fibres for any given
diameter was significantly higher for slow than fast muscle fibres
(F1,878=3968.0; P<0.0001). The relationship
between myonuclei content and fibre diameter is illustrated in
Fig. 11C. Second order
polynomials were fitted to the data (Fig.
11C). For fibres of 60 µm diameter, the myonuclei contents
calculated from the regressions were twofold higher for slow muscle fibres
(2264 nuclei cm–1) than for fast muscle fibres (1126 nuclei
cm–1).
|
Myogenic progenitor cell density
Myogenic progenitor cells (MPCs) were identified using a specific antibody
to Pax 7 (Fig. 12A). Those
MPCs that were committed to differentiation were identified using antibodies
to Fork head protein K1- (FoxK1-;
Fig. 12B), MyoD and Myf-5 (not
illustrated). MPCs immunoreactive to the myogenic regulatory factor Myf-5 were
also quantified. The density of myonuclei
(Fig. 13A) and the density of
Pax 7 immunoreactive cells (Fig.
13B) decreased with increasing fork length. However, in the case
of Pax 7, FL only accounted for 17% of the total variation
(Fig. 13B). The density of
cells immunopositive for the other transcription factors investigated was
similar over the length range 18–48 cm
(Fig. 14A–C). The
average density of cells staining for phenotypic markers of MPCs as a
percentage of total myonuclei density was 3.2% for Pax 7, 2.8% for
FoxK1-, 1.9% for MyoD and 1.7% for Myf-5.
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Rearing experiments
Embryos from the large benthic, pelagic and dwarf benthic morphs were
incubated under two temperature regimes designed to simulate groundwater-fed
spawning sites (2.2–3.2°C) and the general lake temperature
(September–April) (4–7°C). None of the progeny of DB morphs
incubated at the higher temperature survived until hatching. The
log10(fast fibre number) plotted against FL for fish
between hatching and 2–3 weeks after first feeding is illustrated in
Fig. 15A. An ANOVA with morph
and temperature regime as fixed factors revealed no significant differences
between rearing regimes. A linear regression equation, with a slope of 0.19
± 0.017 and an intercept of 2.98 ± 0.051, was fitted to the data
(Fig. 14A;
F1,51=125.1; P<0.0001). The average fibre
diameter also showed no significant differences with rearing temperature
(Fig. 15B). The density of
cells immunopositive for Pax 7, FoxK1-, MyoD and Myf-5 in the offspring
of LB and PL morphs is illustrated in Fig.
16A–C. An ANOVA with rearing temperature and morph as fixed
factors revealed no significant differences between groups. The density of
cells expressing these MPC markers was generally higher than in larger fish
(see Figs 12,
13).
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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). In contrast, most studies have shown that the recruitment
of fast muscle fibres continues until a defined body length, after which
growth is entirely by the hypertrophy of fibres formed earlier in development
(Weatherley et al., 1980,
1988;
Stickland, 1983; Johnston et
al., 2000c,
2003a,c,d).
Postembryonic myogenesis therefore differed between slow and fast muscles with
respect to the duration of fibre recruitment. Skeletal muscle fibres are
post-mitotic and growth involves a population of myogenic progenitor cells
(MPCs) that retain the ability to divide. In mammals, there is considerable
evidence for heterogeneity in the MPCs from different muscle types in adult
stages (Feldman and Stockdale,
1991; Hawke and Garry,
2001; Levin et al.,
2001; Mootoosamy and Dietrich,
2002; Tajbakhsh,
2003). For example, MPCs isolated from adult rabbit fast- and
slow-twitch muscle differentially express the secreted frizzle-related protein
thought to be involved in cell fate determination
(Levin et al., 2001). The
embryological origin and the phenotypes of MPCs in teleost fast and slow
muscles are important questions for future research.
The major finding of the present study was that the recruitment of fast
muscle fibres in the Arctic charr morphs in Thingvallavatn was related to
ultimate body size. Our null hypothesis that the number and size distributions
of myotomal muscle was the same in all morphs was therefore rejected for fast
muscle. The cessation of muscle fibre recruitment in Arctic charr was related
to fork length, not age (Fig.
9), with recruitment stopping at 18 cm for the DB-morph,
24–26 cm for the PL morph and around 33–35 cm for the LB and PI
morphs (Figs 5B,
7). During the hyperplastic
phase of growth fast fibre number scaled with
Mb0.52 in the DB and PL morphs and
Mb0.33 in the LB and PI morphs
(Fig. 8). Thus fibre number
(FN) was lower in juvenile DB than LB morphs, although the increase
in FN with increasing body mass was proportionally greater
(Fig. 8). The cessation of
hyperplasia in fast muscle was not related to sex or correlated with the body
length at sexual maturity, which was 7–8 cm in the DB morph and
25–30 cm in the LB morph. The size of the DB morph at maturity may be
close to the lower limit for gonadal development in salmonids
(Myers et al., 1986). The
dwarf morph grows more slowly, but matures earlier than the large benthic
morph, which reaches a larger ultimate size
(Sandlund et al., 1992). The
pattern of growth and maturation in Arctic charr morphs in Thingvallavatn is
consistent with optimisation theory, which predicts that slow growth at an
early age selects for young age at sexual maturity, whereas growth stagnation
at an old age selects for late sexual maturity
(Schaffer and Elson, 1975).
Hyperplasia in fast muscle had stopped in a 4+ year class DB morph, whereas
the youngest LB and PI morphs that had stopped recruiting fibres were the 7+
year-class (Fig. 9). The age at
which the threshold length for cessation of hyperplasia was reached occurred
over a wide range of ages, such that a 12+ year-class LB morph and a 14+
year-class PI morph were found that were still recruiting fast muscle fibres
(Fig. 9).
The origins of different patterns of fibre recruitment
The environmental temperature during early development has been shown to
affect the number of muscle fibres produced at each stage of myogenesis in
salmonids (Stickland et al.,
1988; Johnston et al.,
2000b,
2003c) and other teleosts
(Ayala et al., 2000;
Johnston et al., 1998).
Temperature regimes that result in a higher fibre number have been associated
with a higher density of myogenic progenitor cells (Johnston et al.,
2000a,
2003c). The effect of this
developmental plasticity varies with the temperature profile during
embryogenesis and can show marked differences between species and between
populations of the same species (Johnston et al.,
2000a,b;
Johnston and Hall, 2004). In
Thingvallavatn, spawning sites that are associated with groundwater input have
a stable temperature of 2–3°C whereas the temperature in other areas
is more seasonally variable
(Skúlason et al.,
1989a; Sandlund et al.,
1992). Both the DB morph and the LB morph spawn in sites affected
by subterranean springs and therefore developmental plasticity is unlikely to
be an explanation for the differences in fibre number between these morphs.
This was confirmed by rearing the progeny of the DB, LB and PL morphs at
temperatures that simulated the temperature in groundwater influenced sites
and the general lake temperature over the period of embryonic development
(September–April; Skúlason et
al., 1989a). No evidence was found for developmental plasticity in
either MPC density or fibre number in the progeny of Arctic charr from
Thingvallavatn (Figs 14A,C,
15A). Furthermore, the
juvenile and adult stages of the DB and LB morphs coexist, and both feed on
snails and other benthic invertebrates
(Sandlund et al., 1992). We
therefore conclude that the differences in fibre recruitment patterns between
morphs probably have a genetic basis. Consistent with this hypothesis,
differences in maximum body size and growth patterns between morphs are
maintained in common garden experiments, indicating a large genetic component
(Skúlason et al.,
1989b,
1993,
1996;
Eiríksson et al.,
1999).
It has been suggested that in the absence of interspecific competition,
pioneer species such as Arctic charr, were subject to intense intraspecific
competition for resources, accompanied by character release related to
phenotypic plasticity in behaviour and life history
(Nordeng, 1983;
Skúlason et al., 1999;
Snorrason and Skúlason,
2004). Habitat diversity would then be expected to facilitate the
diversification of phenotype resulting in discrete resource morphs
(Snorrason and Skúlason,
2004). Given sufficient genetic variation, such polymorphisms can
be consolidated by genetic selection, and possible mechanisms for the origin
of reproductive isolation have been proposed (see
Snorrason and Skúlason,
2004). Only the smallest charr would be able to exploit the
interstitial spaces of the stony substrate of the lake and size-assortative
mating might be expected to promote isolation, restrict gene flow with larger
charr and in time lead to the formation of a genetically stable dwarf. There
is evidence that such isolation processes can progress very rapidly in the
wild in salmonids (Hendry et al.,
2000). The Arctic charr morphs in Thingvallavatn are generally
considered to have a sympatric origin
(Snorrason et al., 1994;
Skúlason et al., 1999),
a view supported to varying degrees by data on the genetic structuring of
populations within and between lakes
(Gíslason et al., 1999;
Wilson et al., 2004). An
alternative view is that the charr constitute a species complex that evolved
allopatrically during the Pleistocene glaciations
(Nyman et al., 1981). In
either case, the morphs in Thingvallavatn have been diverging for no longer
than 10 000 years. Assuming our hypothesis of a genetic explanation is correct
then the genes regulating fibre number and the duration of fibre recruitment
in fast muscle are under high selection pressure and susceptible to rapid
evolutionary change.
The optimal fibre size hypothesis
We propose an `optimal fibre size hypothesis' to explain evolutionary
adjustments in muscle fibre number with body size and temperature. The
hypothesis requires that there is an optimal maximum fibre diameter, which
reflects a trade-off between avoiding diffusional constraints and the need to
minimise the costs of ion pumping. Maintenance of ionic homeostasis is thought
to constitute 20–40% of the resting metabolic rate in teleosts
(Jobling, 1994). The fast
myotomal muscle comprises more than 60% of body mass in a typical teleost, and
therefore ionic homeostasis in this tissue makes an important contribution to
the overall metabolic rate. The surface/volume ratio of muscle fibres
decreases with increasing fibre diameter. Thus the surface area for passive
membrane leak in large diameter fibres would be expected to be less than for
small diameter fibres, and therefore require concomitantly fewer active ion
pumps per unit volume in order to maintain ionic equilibria and membrane
potential. The Na+-K+ pump and the L-type
Ca2+ pump are probably the most important energy consuming pumps in
the muscle sarcolemmal membrane (Clausen,
2003). Important and testable predictions of the hypothesis are
that the number of these pumps per unit volume of muscle and their
contribution to the oxygen consumption of fibres should decrease with
increasing fibre size. Factors such as temperature that change the oxygen
concentration at the surface of the muscle fibre and metabolic demand would be
expected to shift the optimum diameter and hence the number of muscle fibres
required to reach a particular body size. Juvenile and adult Arctic charr in
Thingvallavatn live at the same temperature and the similar fibre type
proportions and myonuclear content of fibres suggest similar activity
patterns. In this case, selection for a small body size in the dwarf morph
would have resulted in a concomitant reduction in FNmax to
produce the optimal trade-off between fibre size and the costs of ionic
homeostasis.
Scaling of maximum fibre diameter
For fish greater than 10 g, the maximum diameter was related to
Mb by the following equation:
Dmax=aMbb where a and b are
constants. Double logarithmic plots of Dmax vs
Mb gave a scaling coefficient (b) of 0.18
(Fig. 10). Smaller fish
contained larger diameter fibres than predicted by the equations in
Fig. 10. In the fast muscle,
plotting the data for fish between 100 and 1893 g resulted in a slope of 0.22
without a significant decrease in the correlation coefficient. A. V. Hill
provided a solution for the maximum radius (RO) that
oxygen can penetrate in a long circular cylinder of muscle for a given oxygen
concentration at its surface (YO):
 | (1) |
). The
mass-specific oxygen consumption at the whole animal level scales with
Mb–0.22 in salmonids
(Clarke and Johnston, 1999).
As body size increases the decrease in mass-specific metabolic rate may relax
diffusional constraints (i.e. the largest diameter of fibre that is permitted
without forming an anoxic core), setting Dmax at a higher
value. Even in the largest fish the diameter of the majority of fibres is much
less than Dmax (Fig.
5A). Therefore somatic growth largely reflects the hypertrophy of
fibres that are sub-maximal in diameter.
In vivo the situation is much more complex than described in
Equation 1 since the oxygen
concentration at the surface of the muscle fibre will depend on the complex
geometry of the capillaries supplying the muscle fibres, the perfusion rates
of individual capillaries, hematocrit, and the numerous factors influencing
the gradient of oxygen between the capillary wall and the mitochondria
(Salathe and Gorman, 1997).
These factors include the myoglobin concentration, the density of lipid-rich
subcellular structures (mitochondria and sarcoplasmic reticulum), and the
distribution of the sites of oxygen consumption as well as the metabolic
demand (Eggleton et al., 2000;
Salathe and Gorman, 1997;
Sidell, 1998).
There are several lines of evidence to suggest that diffusion of oxygen
limits the maximum diameter of the fast muscle fibres even though contraction
is supported from local energy stores and involves anaerobic metabolic
pathways. The immediate energy supply for contraction in the fast muscle comes
from the hydrolysis of phosphocreatine and is supplemented by anaerobic
glycogenolysis during prolonged high-speed swimming, resulting in the
accumulation of lactic acid (Hochachka,
1985; Richards et al.,
2002). The rate of radial diffusion of a substance is proportional
to the reciprocal of the square root of the relative molecular mass, and is
much greater for relatively large molecules such as lactate than oxygen
(Kinsey and Moerland, 2002).
However, in the rainbow trout Oncorhyncus mykiss, the sarcolemmal of
fast muscle fibres is relatively impermeable to lactate
(Sharpe and Milligan, 2003),
resulting in low rates of lactate efflux to the circulation during the
recovery period (Milligan and Wood,
1986). Instead, lactate is retained within the muscle after
exercise and used as a substrate for in situ glycogen resynthesis
(Kieffler, 2000). Ultimately recovery of metabolism to the resting condition
is dependent on oxidative phosphorylation. Although sites of SDHase
localisation were more abundant in the periphery of fast muscle fibres in
Arctic charr, some staining was observed in the central core, consistent with
the presence of mitochondria (not shown). An ultrastructural study in the
brook trout Salvelinus fontinalis also found mitochondria in the core
of the largest diameter fast muscle fibres
(Johnston and Moon, 1981).
Thus, the maximum diameter of fast muscle fibres may well be limited by oxygen
diffusion during recovery metabolism, reflecting the need to sustain these
central mitochondria.
Myogenic progenitor cells and fibre recruitment
Myogenic regulatory factors (MRFs
