Coho salmon ( Oncorhynchus kisutch ) transgenic for growth hormone (GH) show substantially faster growth than wild-type (WT) fish. We fed GH-transgenic salmon either to satiation (1 year; TF) or the same smaller ration of wild-type fish (2 years; TR), resulting in groups matched for body size to WT salmon. The myotomes of TF and WT fish had the same number and size distribution of muscle fibres, indicating a twofold higher rate of fibre recruitment in the GH transgenics. Unexpectedly, calorie restriction was found to decrease the rate of fibre production in transgenics, resulting in a 20% increase in average fibre size and reduced costs of ionic homeostasis. Genes for myotube formation were downregulated in TR relative to TF and WT fish. We suggest that muscle fibre size optimisation allows the reallocation of energy from maintenance to locomotion, explaining the observation that calorie-restricted transgenics grow at the same rate as WT fish whilst exhibiting markedly higher foraging activity.

SUMMARY The short generation time of the zebrafish ( Danio rerio ) was exploited to investigate the effects of selection for body size at age on early life-history traits and on the transcriptional response to a growth stimulus in skeletal muscle of adult fish. Replicate populations were either unselected (U-lineage) or subjected to four generations of experimental selection for small (S-lineage) or large (L-lineage) body size at 90 days post-fertilization. Body mass was on average 16.3% and 41.0% higher in the L- than in the U- and S-lineages, respectively. Egg diameter was 6.4% lower with 13% less yolk in the S-lineage compared with the other lineages. Maternal transcripts for igf2r , bmpr1aa , igf1ar , igf2a , igfbp5a , ghra and igfbp3 in 2–4 cell stage embryos were higher in the L- than in the S-lineage. Larvae from the L-lineage were significantly larger, but survivorship at the end of the first month was similar between lineages. Gene expression was measured in the fast muscle of adult fish fasted for 7 days and then re-fed to satiation for 48 h. The expression of 11 insulin-like growth factor pathway genes and 12 other nutritionally responsive genes was similar for the S- and L-lineages as was gut fullness with feeding. Transcript abundance for four genes ( igf1a , igf2r , igfbp1a and igfbp1b ) showed either regulated or constitutive differences between the S- and L-lineages. For example, igf2 receptor transcript abundance was higher and igbp1a/b transcript abundance was lower in the L- than in the S-lineage, consistent with an effect of selection on insulin-like growth factor signalling.

SUMMARY Male zebrafish ( Danio rerio ) were fasted for 7 days and fed to satiation over 3 h to investigate the transcriptional responses to a single meal. The intestinal content at satiety (6.3% body mass) decreased by 50% at 3 h and 95% at 9 h following food withdrawal. Phosphorylation of the insulin-like growth factor (IGF) signalling protein Akt peaked within 3 h of feeding and was highly correlated with gut fullness. Retained paralogues of IGF hormones genes were regulated with feeding, with igf1a showing a pronounced peak in expression after 3 h and igf2b after 6 h. Igf-I receptor transcripts were markedly elevated with fasting, and decreased to their lowest levels 45 min after feeding. igf1rb transcripts increased more quickly than igf1ra transcripts as the gut emptied. Paralogues of the insulin-like growth factor binding proteins (IGFBPs) were constitutively expressed, except for igfbp1a and igfbp1b transcripts, which were significantly elevated with fasting. Genome-wide transcriptional responses were analysed using the Agilent 44K oligonucleotide microarray and selected genes validated by qPCR. Fasting was associated with the upregulation of genes for the ubiquitin-proteasome degradation pathway, anti-proliferative and pro-apoptotic genes. Protein chaperones ( unc45b, hspd1, hspa5, hsp90a.1, hsp90a.2 ) and chaperone interacting proteins ( ahsa1 and stip1 ) were upregulated 3 h after feeding along with genes for the initiation of protein synthesis and mRNA processing. Transcripts for the enzyme ornithine decarboxylase 1 showed the largest increase with feeding (11.5-fold) and were positively correlated with gut fullness. This study demonstrates the fast nature of the transcriptional responses to a meal and provides evidence for differential regulation of retained paralogues of IGF signalling pathway genes.

Summary Teleost muscle first arises in early embryonic life and its development is driven by molecules present in the egg yolk and modulated by environmental stimuli including temperature and oxygen. Several populations of myogenic precursor cells reside in the embryonic somite and external cell layer and contribute to muscle fibres in embryo, larval, juvenile and adult stages. Many signalling proteins and transcription factors essential for these events are known. In all cases, myogenesis involves myoblast proliferation, migration, fusion and terminal differentiation. Maturation of the embryonic muscle is associated with motor innervation and the development of a scaffold of connective tissue and complex myotomal architecture needed to generate swimming behaviour. Adult muscle is a heterogeneous tissue composed of several cell types that interact to affect growth patterns. The development of capillary and lymphatic circulations and extramuscular organs – notably the gastrointestinal, endocrine, neuroendocrine and immune systems – serves to increase information exchange between tissues and with the external environment, adding to the complexity of growth regulation. Teleosts often exhibit an indeterminate growth pattern, with body size and muscle mass increasing until mortality or senescence occurs. The dramatic increase in myotomal muscle mass between embryo and adult requires the continuous production of muscle fibres until 40–50% of the maximum body length is reached. Sarcomeric proteins can be mobilised as a source of amino acids for energy metabolism by other tissues and for gonad generation, requiring the dynamic regulation of muscle mass throughout the life cycle. The metabolic and contractile phenotypes of muscle fibres also show significant plasticity with respect to environmental conditions, migration and spawning. Many genes regulating muscle growth are found as multiple copies as a result of paralogue retention following whole-genome duplication events in teleost lineages. The extent to which indeterminate growth, ectothermy and paralogue preservation have resulted in modifications of the genetic pathways regulating muscle growth in teleosts compared to mammals largely remains unknown. This review describes the use of compensatory growth models, transgenesis and tissue culture to explore the mechanisms of muscle growth in teleosts and provides some perspectives on future research directions.

SUMMARY We investigated the effects of embryonic temperature (ET) treatments (22,26 and 31°C) on the life-time recruitment of fast myotomal muscle fibres in zebrafish Danio rerio L. reared at 26/27°C from hatching. Fast muscle fibres were produced until 25 mm total length (TL) at 22°C ET, 28 mm TL at 26°C ET and 23 mm TL at 31°C ET. The final fibre number (FFN)showed an optimum at 26°C ET (3600) and was 19% and 14% higher than for the 22°C ET (3000) and 31°C ET (3100) treatments, respectively. Further growth to the maximum TL of ∼48 mm only involved fibre hypertrophy. Microarray experiments were used to determine global changes in microRNA (miRNA) and mRNA expression associated with the transition from the hyperplasic myotube-producing phenotype (M + , 10–12 mm TL) to the hypertrophic growth phenotype (M – , 28–31 mm TL) in fish reared at 26–27°C over the whole life-cycle. The expression of miRNAs and mRNAs obtained from microarray experiments was validated by northern blotting and real-time qPCR in independent samples of fish with the M + and M – phenotype. Fourteen down-regulated and 15 up-regulated miRNAs were identified in the M – phenotype together with 34 down-regulated and 30 up-regulated mRNAs (>2-fold; P <0.05). The two most abundant categories of down-regulated genes in the M – phenotype encoded contractile proteins (23.5%) and sarcomeric structural/cytoskeletal proteins (14.7%). In contrast, the most highly represented up-regulated transcripts in the M – phenotype were energy metabolism (26.7%) and immune-related (20.0%) genes. The latter were mostly involved in cell–cell interactions and cytokine pathways and included β-2-microglobulin precursor ( b2m ), an orthologue of complement component 4, invariant chain-like protein 1( iclp ), CD9 antigen-like ( cd9l ), and tyrosine kinase,non-receptor ( tnk2 ). Five myosin heavy chain genes that were down-regulated in the M – phenotype formed part of a tandem repeat on chromosome 5 and were shown by in situ hybridisation to be specifically expressed in nascent myofibres. Seven up-regulated miRNAs in the M – phenotype showed reciprocal expression with seven mRNA targets identified in miRBase Targets version 5( http://microrna.sanger.ac.uk/targets/v5/ ),including asporin (aspn) which was the target for four miRNAs. Eleven down-regulated miRNAs in the M – phenotype had predicted targets for seven up-regulated genes, including dre-miR-181c which had five predicted mRNA targets. These results provide evidence that miRNAs play a role in regulating the transition from the M + to the M – phenotype and identify some of the genes and regulatory interactions involved.

SUMMARY In this study we describe the complete coding sequence for insulin-like growth factor I (IGF-I), insulin-like growth factor II (IGF-II), insulin-like growth factor binding protein (IGFBP) 1, 2, 4, 5 and 6 and IGFBP-related protein 1 (IGFBP-rP1) of Atlantic salmon ( Salmo salar L.). We also report the characterisation of two gene paralogues of IGFBP-2 and IGFBP-5. Following 22 days restricted feeding (0 d) to achieve zero growth, fish were fed to satiation and sampled at 3, 5, 7, 14, 30 and 60 days. Expression profiles for genes involved in the IGF signalling pathway in fast myotomal muscle were determined using real-time quantitative RT-PCR. The transition from zero to fast growth is characterised by constitutive upregulation of IGF-I and IGFBP-4, a transient increase in IGFBP-5.2, and downregulation of IGFBP-2.1, IGF-II, IGF2R (IGF-II receptor) and IGFR1a (IGF-I receptor a). Expression of IGFBP-2.2, IGFBP-5.1, IGFBP-6, IGFBP-rP1 and IGFR1b showed little or no response to feeding. Expression of the myogenic marker genes myogenin, MHC and MLC2 were higher with feed restriction, and decreased as an early response to feeding, before increasing to a peak at 14 days,corresponding with a peak in IGF-I expression. IGFBP-4, which contains a putative connective tissue localisation signal, was the only IGFBP constitutively upregulated following feeding, and was positively correlated with IGF-I expression. Together, these data show that switching to fast growth in Atlantic salmon skeletal muscle involves the local upregulation of IGF-I,IGFBP-5.2 and IGFBP-4, with downregulation of IGFBP-2.1.

SUMMARY FoxK1 is a member of the highly conserved forkhead/winged helix (Fox)family of transcription factors and it is known to play a key role in mammalian muscle development and myogenic stem cell function. The tiger pufferfish ( Takifugu rubripes ) orthologue of mammalian FoxK1 ( TFoxK1 ) has seven exons and is located in a region of conserved synteny between pufferfish and mouse. TFoxK1 is expressed as three alternative transcripts: TFoxK1-α, TFoxK1-γ and TFoxK1-δ. TFoxK1-α is the orthologue of mouse FoxK1-α , coding for a putative protein of 558 residues that contains the forkhead and forkhead-associated domains typical of Fox proteins and shares 53% global identity with its mammalian homologue. TFoxK1-γ and TFoxK1-δ arise from intron retention events and these transcripts translate into the same 344-amino acid protein with a truncated forkhead domain. Neither are orthologues of mouse FoxK1-β . In adult fish, the TFoxK1 splice variants were differentially expressed between fast and slow myotomal muscle, as well as other tissues, and the FoxK1-α protein was expressed in myogenic progenitor cells of fast myotomal muscle. During embryonic development, TFoxK1 was transiently expressed in the developing somites, heart,brain and eye. The relative expression of TFoxK1-α and the other two alternative transcripts varied with the incubation temperature regime for equivalent embryonic stages and the differences were particularly marked at later developmental stages. The developmental expression pattern of TFoxK1 and its localisation to mononuclear myogenic progenitor cells in adult fast muscle indicate that it may play an essential role in myogenesis in T. rubripes .

SUMMARY Potential molecular mechanisms regulating developmental plasticity to temperature were investigated in Atlantic salmon embryos ( Salmo salar L.). Six orthologues of the four myogenic regulatory factors (MRFs:individually: smyf5, smyoD1a/1b/1c, smyoG and sMRF4), the master transcription factors regulating vertebrate myogenesis, were characterised at the mRNA/genomic level. In situ hybridisation was performed with specific cRNA probes to determine the expression patterns of each gene during embryonic myogenesis. To place the MRF data in the context of known muscle fibre differentiation events, the expression of slow myosin light chain-1 and Pax7 were also investigated. Adaxial myoblasts expressed smyoD1a prior to and during somitogenesis followed by smyoD1c (20-somite stage, ss),and sMRF4 (25–30 ss), before spreading laterally across the myotome, followed closely by the adaxial cells. Smyf5 was detected prior to somitogenesis, but not in the adaxial cells in contrast to other teleosts studied. The expression domains of smyf5 , smyoD1b and smyoG were not confined to the s-smlc1 expression field,indicating a role in fast muscle myogenesis. From the end of segmentation,each MRF was expressed to a greater or lesser extent in zones of new muscle fibre production, the precursor cells for which probably originated from the Pax7 expressing cell layer external to the single layer of s-smlc1 + fibres. SmyoD1a and smyoG showed similar expression patterns with respect to somite stage at three different temperatures investigated (2°C, 5°C and 8°C) in spite of different rates of somite formation (one somite added each 5 h, 8 h and 15 h at 8°C, 5°C and 2°C, respectively). In contrast, the expression of smyf5 , sMRF4 and s-smlc1 was retarded with respect to somite stage at 2°C compared to 8°C, potentially resulting in heterochronies in downstream pathways influencing later muscle phenotype.

SUMMARY Embryonic development in teleosts is profoundly affected by environmental conditions, particularly temperature and dissolved oxygen concentrations. The environment determines the rate of myogenesis, the composition of sub-cellular organelles, patterns of gene expression, and the number and size distribution of muscle fibres. During the embryonic and larval stages, muscle plasticity to the environment is usually irreversible due to the rapid pace of ontogenetic change. In the early life stages, muscle can affect locomotory performance and behaviour, with potential consequences for larval survival. Postembryonic growth involves myogenic progenitor cells (MPCs) that originate in the embryo. The embryonic temperature regime can have long-term consequences for the growth of skeletal muscle in some species, including the duration and intensity of myotube formation in adult stages. In juvenile and adult fish,abiotic (temperature, day-length, water flow characteristics, hypoxia) and biotic factors (food availability, parasitic infection) have complex effects on the signalling pathways regulating the proliferation and differentiation of MPCs, protein synthesis and degradation, and patterns of gene expression. The phenotypic responses observed to the environment frequently vary during ontogeny and are integrated with endogenous physiological rhythms,particularly sexual maturation. Studies with model teleosts provide opportunities for investigating the underlying genetic mechanisms of muscle plasticity that can subsequently be applied to non-model species of more ecological or commercial interest.

SUMMARY Three embryonic class II myosin heavy chains (MYHs) were cloned from the common carp ( Cyprinus carpio L.), MYH emb1 ,MYH emb2 and MYH emb3 . MYH DNA clones were also isolated from the slow muscle of adult carp acclimated to 10°C (MYH S10 )and 30°C (MYH S30 ). Phylogenetic analysis demonstrated that MYH emb1 and MYH emb2 belonged to the fast skeletal muscle MYH clade. By contrast, the sequence of MYH emb3 was similar to the adult slow muscle isoforms, MYH S10 and MYH S30 . MYH emb1 and MYH emb2 transcripts were first detected by northern blot analysis in embryos 61 h post-fertilization (h.p.f.) at the heartbeat stage, with peak expression occurring in 1-month-old juveniles. MYH emb1 continued to be expressed at low levels in 7-month-old juveniles when MYH emb2 was not detectable. MYH emb3 transcripts appeared at almost the same stage as MYH emb1 transcripts did (61 h.p.f.), and these genes showed a similar pattern of expression. Whole mount in situ hybridization analysis revealed that the transcripts of MYH emb1 and MYH emb2 were expressed in the inner part of myotome, whereas MYH emb3 was expressed in the superficial compartment. MYH S10 and MYH S30 mRNAs were first detected at hatching. In adult stages, the expression of slow muscle MYH mRNAs was dependent on acclimation temperature. MYH S10 mRNA was expressed at an acclimation temperature of 10 and 20°C, but not at 30°C. In contrast, MYH S30 mRNA was strongly expressed at all acclimation temperatures. The predominant MYH transcripts found in adult slow muscle and in embryos at hatching were expressed in adult fast muscle at some acclimation temperatures but not others. A MYH DNA clone was isolated from the cardiac muscle of 10°C-acclimated adult fish (MYH card ). MYH card mRNA was first detected at 61 h.p.f., but strong signals were only observed in the adult myocardium. The present study has therefore revealed a complex pattern of expression of MYH genes in relation to developmental stage, muscle type and acclimation temperature. None of the skeletal muscle MYHs identified so far was strongly expressed during the late juvenile stage, indicating further developmentally regulated members of the MYH II gene family remain to be discovered.

SUMMARY Thingvallavatn, the largest and one of the oldest lakes in Iceland,contains four morphs of Arctic charr Salvelinus alpinus . Dwarf benthic (DB), large benthic (LB), planktivorous (PL) and piscivorous (PI)morphs can be distinguished and differ markedly in head morphology,colouration and maximum fork length ( FL max ), reflecting their different resource specialisations within the lake. The four morphs in Thingvallavatn are thought to have been isolated for approximately 10 000 years, since shortly after the end of the last Ice Age. 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( M b ) 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 M b 0.45 . 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( FN max ) in the dwarf morph (6.97×10 4 ) was 56.5% of that found in the LB and PI morphs combined(1.23×10 5 ) ( 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-α (FoxK1-α), 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 FN max 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.

SUMMARY Embryos of the common carp, Cyprinus carpio L., were reared from fertilization of the eggs to inflation of the swim bladder in the larval stage at 18 and 25°C. cRNA probes were used to detect transcripts of the myogenic regulatory factors MyoD, Myf-5 and myogenin, and five myosin heavy chain (MyHC) isoforms during development. The genes encoding Myf-5 and MyoD were switched on first in the unsegmented mesoderm, followed by myogenin as the somites developed. Myf -5 and MyoD transcripts were initially limited to the adaxial cells, but Myf -5 expression spread laterally into the presomitic mesoderm before somite formation. Two distinct bands of staining could be seen corresponding to the cellular fields of the forming somites, but as each furrow delineated, Myf-5 mRNA levels declined. Upon somite formation, MyoD expression spread laterally to encompass the full somite width. Expression of the myogenin gene was also switched on during somite formation, and expression of both transcripts persisted until the somites became chevron-shaped. Expression of MyoD was then downregulated shortly before myogenin . The expression patterns of the carp myogenic regulatory factor (MRF) genes most-closely resembled that seen in the zebrafish rather than the rainbow trout (where expression of MyoD remains restricted to the adaxial domain of the somite for a prolonged period) or the herring (where expression of MyoD persists longer than that of myogenin ). Expression of two embryonic forms of MyHC began simultaneously at the 25-30 somite stage and continued until approximately two weeks post-hatch. However, the three adult isoforms of fast muscle MyHC were not detected in any stage examined, emphasizing a developmental gap that must be filled by other, as yet uncharacterised, MyHC isoform(s). No differences in the timing of expression of any mRNA transcripts were seen between temperature groups. A phylogenetic analysis of the MRFs was conducted using all available full-length amino acid sequences. A neighbour-joining tree indicated that all four members evolved from a common ancestral gene, which first duplicated into two lineages, each of which underwent a further duplication to produce Myf-5 and MyoD , and myogenin and MRF4 . Parologous copies of MyoD from trout and Xenopus clustered closely together within clades, indicating recent duplications. By contrast, MyoD paralogues from gilthead seabream were more divergent, indicating a more-ancient duplication.

SUMMARY Atlantic salmon ( Salmo salar L.) were fed to satiety and reared from ∼60 g to 5000 g at ambient seawater temperatures. The effect of photoperiod manipulation on muscle growth was investigated from the start of the first sea winter. Continuous light treatment in winter/spring (1 November to 18 June) improved growth performance in fish, resulting in a 30% increase in mean body mass relative to the ambient photoperiod fish by 12 August, but had no effect on sexual maturation. Significant increases in body mass in the continuous light groups were observed after 126 days ( P <0.01). The number of fast muscle fibres per trunk cross-section was determined in a subset of the fish and was 28.5% higher in the continuous light(799×10 3 ) than the natural day length(644×10 3 ) groups after only 40 days, corresponding to the period of decreasing natural day length. Subsequent rates of fibre recruitment were similar between treatments. At the end of the fibre recruitment phase of growth (combined June and August samples), the maximum number of fast muscle fibres was 23% higher in fish from the cages receiving continuous light(881×10 3 ±32×10 3 ; N =19) than in the ambient photoperiod cages(717×10 3 ±15×10 3 ; N =20)( P <0.001). Continuous light treatment was associated with a shift in the distribution of fibre diameters, reflecting the altered patterns of fibre recruitment. However, the mean rate of fibre hypertrophy showed no consistent difference between treatments. There was a linear relationship between the myonuclear content of isolated single fibres and fibre diameter. On average, there were 27% more myonuclei in 150 μm-diameter fibres in the continuous light (3118 myonuclei cm -1 ) than the ambient photoperiod(2448 myonuclei cm -1 ) fish. After 40 days, continuous light treatment resulted in a transient increase in the density of myogenic progenitor cells, identified using a c-met antibody, to a level 70% above that of fish exposed to natural light. It is suggested that short days inhibited the proliferation of myogenic progenitor cells and that this was overcome by transferring fish to continuous light, causing an increase in the number of times the myogenic precursor cells divided and/or a decrease in cell cycle time. The net increase in myogenic progenitor cells resulted in proportional increases in the number and myonuclear content of fibres. The subsequent hypertrophy of these additional fibres can explain the delayed increase in body mass observed with continuous light treatment.

SUMMARY Seven cDNA clones coding for different muscle-specific proteins (MSPs) were isolated from the fast muscle tissue of Atlantic cod Gadus morhua L. In situ hybridization using cRNA probes was used to characterize the temporal and spatial patterns of gene expression with respect to somite stage in embryos incubated at 4°C, 7°C and 10°C. MyoD transcripts were first observed in the presomitic mesoderm prior to somite formation, and in the lateral compartment of the forming somites. MyoD expression was not observed in the adaxial cells that give rise to the slow muscle layer, and expression was undetectable by in situ hybridization in the lateral somitic mesoderm after the 35-somite stage,during development of the final ∼15 somites. RT-PCR analysis, however,confirmed the presence of low levels of the transcript during these later stages. A phylogenetic comparison of the deduced aminoacid sequences of the full-length MyoD cDNA clone and those from other teleosts, and inference from the in situ expression pattern suggested homology with a second paralogue ( MyoD2 ) recently isolated from the gilthead seabream Sparus aurata . Following MyoD expression,α- actin was the first structural gene to be switched on at the 16-somite stage, followed by myosin heavy chain, troponin T, troponin I and muscle creatine kinase. The final mRNA in the series to be expressed was troponin C . All genes were switched on prior to myofibril assembly. The troponin C sequence was unusual in that it showed the greatest sequence identity with the rainbow trout Oncorhynchus mykiss cardiac/slow form, but was expressed in the fast myotomal muscle and not in the heart. In addition, the third TnC calcium binding site showed a lower level of sequence conservation than the rest of the sequence. No differences were seen in the timing of appearance or rate of posterior progression (relative to somite stage) of any MSP transcripts between embryos raised at the different temperatures. It was concluded that myofibrillar genes are activated asynchronously in a distinct temporal order prior to myofibrillar assembly and that this process was highly canalized over the temperature range studied.

SUMMARY The fish fauna of the continental shelf of the Southern Ocean is dominated by a single sub-order of Perciformes, the Notothenioidei, which have unusually large diameter skeletal muscle fibres. We tested the hypothesis that in fast myotomal muscle a high maximum fibre diameter ( FD max ) was related to a reduction in the number of muscle fibres present at the end of the recruitment phase of growth. We also hypothesized that the maximum fibre number ( FN max ) would be negatively related to body size,and that both body size and size-corrected FN max would show phylogenetic signal (tendency for related species to resemble each other). Finally, we estimated ancestral values for body size and FN max . A molecular phylogeny was constructed using 12S mitochondrial rRNA sequences. A total of 16 species were studied from the Beagle Channel, Tierra del Fuego (5-11°C), Shag Rocks, South Georgia(0.5-4°C), and Adelaide Island, Antarctic Peninsula (-1.5 to 0.5°C). The absence of muscle fibres of less than 10 μm diameter was used as the criterion for the cessation of fibre recruitment. FD max increased linearly with standard length ( SL ), reaching 500-650 μm in most species. Maximum body size was a highly significant predictor of species variation in FN max , and both body size and size-corrected FN max showed highly significant phylogenetic signal ( P <0.001). Estimates of trait values at nodes of the maximum likelihood phylogenetic tree were consistent with a progressive reduction in fibre number during part of the notothenioid radiation, perhaps serving to reduce basal energy requirements to compensate for the additional energetic costs of antifreeze production. For example, FN max in Chaenocephalus aceratus (12 700±300, mean ± s . e . m ., N =18) was only 7.7% of the value found in Eleginops maclovinus (164 000±4100, N =17), which reaches a similar maximum length (85 cm). Postembryonic muscle fibre recruitment in teleost fish normally involves stratified followed by mosaic hyperplasia. No evidence for this final phase of growth was found in two of the most derived families(Channichthyidae and Harpagiferidae). The divergence of the notothenioids in Antarctica after the formation of the Antarctic Polar Front and more recent dispersal north would explain the high maximum diameter and low fibre number in the derived sub-Antarctic notothenioids. These characteristics of notothenioids may well restrict their upper thermal tolerance, particularly for Champsocephalus esox and similar Channichthyids that lack respiratory pigments.

SUMMARY 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 (α) and the instantaneous growth rate per unit change in temperature (γ). 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 age×treatment 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 (9.3×10 5 ±2.0×10 4 than in the heated (7.6×10 5 ±1.5×10 4 )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 age×treatment 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.

SUMMARY In teleosts, the proliferation of myogenic progenitor cells is required for muscle growth and nuclear turnover. We measured the cell cycle and S-phase duration of myogenic cells in the fast myotomal muscle of two closely related Harpagifer species by cumulative S-phase labelling with 5-bromo-2′-deoxyuridine (BrdU). Harpagifer antarcticus is a stenothermal species from the Antarctic peninsula (experiencing temperatures of -2°C to +1°C) and Harpagifer bispinis is a eurythermal species from the Beagle Channel, Tierra del Fuego (living at +4°C in winter and up to 11°C in summer). Specific growth rates in the adult stages studied were not significantly different from zero. Myogenic progenitor cells were identified using an antibody against c-met. Seventy-five percent of the c-met +ve cells were in a proliferative state in both species. Cell cycle time was 150 h at 5°C and 81.3 h at 10°C in H. bispinis (Q 10 =3.4). Cell cycle duration was 35% shorter in H. antarcticus at 0°C (111 h) than in H. bispinis at 5°C. The predicted cell cycle time for H. bispinis at 0°C(based on the Q 10 relationship) was 277 h, which was more than double that measured for the Antarctic species at this temperature. The results obtained are compatible with an evolutionary adjustment of cell cycle time for function at low temperature in the Antarctic species.

SUMMARY Feeding metabolism and the activation of myogenic progenitor cells were investigated in the fast myotomal muscle of the sub-Antarctic fish Hapagifer bispinis acclimatized to either simulated summer (10°C; 18 h:6 h light:dark) or simulated winter (5°C; 6 h:18 h light:dark) conditions. Ingestion of a single meal equivalent to 10% and 15% of body mass in simulated winter and summer groups, respectively, resulted in an average 2.6-fold and 3.6-fold increase in oxygen consumption, declining to 75% of peak values after 63 h and 46 h. In fasted individuals, the number of myogenic progenitor cells, identified by the expression of c-met, was not significantly different between simulated summer and winter fish, representing 6.6% and 5.8% of total myonuclei, respectively. However, the number of cells expressing myogenin was higher whereas the expression of MyoD was lower in winter than in summer groups. The ingestion of a single meal under winter and summer treatment regimes resulted in a significant increase in the number of cells expressing MyoD (51% and 111%) and PCNA (88% and 140%, respectively). This was followed by an increase in the abundance of c-met (74 and 85%) and myogenin (42 and 97%, respectively) positive cells, indicating the production of new myogenic progenitor cells and the commitment to differentiation of a number of them. These results show that the proliferation of myogenic progenitor cells can be induced by feeding in teleost fishes and that temperature and photoperiod influence the expression of myogenic regulatory factors.

SUMMARY Seasonal cooling can modify the thermal preferenda of ectothermic vertebrates and elicit a variety of physiological responses ranging from winter dormancy to an acclimation response that partially compensates for the effects of low temperature on activity. Partial compensation of activity levels is particularly common in aquatic species for which seasonal temperature changes provide a stable cue for initiating the response. Thermal plasticity of locomotory performance has evolved independently on numerous occasions, and there is considerable phylogenetic diversity with respect to the mechanisms at the physiological and molecular levels. In teleosts,neuromuscular variables that can be modified include the duration of motor nerve stimulation, muscle activation and relaxation times, maximum force and unloaded shortening velocity ( V max ), although not all are modified in every species. Thermal plasticity in V max has been associated with changes in myosin ATPase activity and myosin heavy chain(MyHC) composition and/or with a change in the ratio of myosin light chain isoforms. In common carp ( Cyprinus carpio ), there are continuous changes in phenotype with acclimation temperature at lower levels of organisation, such as MyHC composition and V max , but a distinct threshold for an effect in terms of locomotory performance. Thus,there is no simple relationship between whole-animal performance and muscle phenotype. The nature and magnitude of temperature acclimation responses also vary during ontogeny. For example, common carp acquire the ability to modify MyHC composition with changes in acclimation temperature during the juvenile stage. In contrast, the thermal plasticity of swimming performance observed in tadpoles of the frog Limnodynastes peronii is lost in the terrestrial adult stage. Although it is often assumed that the adjustments in locomotory performance associated with temperature acclimation enhance fitness, this has rarely been tested experimentally. Truly integrative studies of temperature acclimation are scarce, and few studies have considered both sensory and motor function in evaluating behavioural responses. Developmental plasticity is a special case of a temperature acclimation response that can lead to temporary or permanent changes in morphology and/or physiological characteristics that affect locomotory performance.

SUMMARY Motor units are the functional units of muscle contraction in vertebrates. Each motor unit comprises muscle fibres of a particular fibre type and can be considered as fast or slow depending on its fibre-type composition. Motor units are typically recruited in a set order, from slow to fast, in response to the force requirements from the muscle. The anatomical separation of fast and slow muscle in fish permits direct recordings from these two fibre types. The frequency spectra from different slow and fast myotomal muscles were measured in the rainbow trout Oncorhynchus mykiss . These two muscle fibre types generated distinct low and high myoelectric frequency bands. The cat paw-shake is an activity that recruits mainly fast muscle. This study showed that the myoelectric signal from the medial gastrocnemius of the cat was concentrated in a high frequency band during paw-shake behaviour. During slow walking, the slow motor units of the medial gastrocnemius are also recruited, and this appeared as increased muscle activity within a low frequency band. Therefore, high and low frequency bands could be distinguished in the myoelectric signals from the cat medial gastrocnemius and probably corresponded, respectively, to fast and slow motor unit recruitment. Myoelectric signals are resolved into time/frequency space using wavelets to demonstrate how patterns of motor unit recruitment can be determined for a range of locomotor activities.