Vera L. A. Vieira

Reduction in muscle fibre number during the adaptive radiation of notothenioid fishes: a phylogenetic perspective

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 (FDmax) 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 (FNmax) would be negatively related to body size, and that both body size and size-corrected FNmax would show phylogenetic signal (tendency for related species to resemble each other). Finally, we estimated ancestral values for body size and FNmax. 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. FDmax 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 FNmax, and both body size and size-corrected FNmax 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, FNmax 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.

Rapid evolution of muscle fibre number in post-glacial populations of Arctic charr Salvelinus alpinus.

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 (FLmax), 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 (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 (6.97×104) was 56.5% of that found in the LB and PI morphs combined (1.23×105) (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 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.

The parallel evolution of dwarfism in Arctic charr is accompanied by adaptive divergence in mTOR-pathway gene expression

Ecological factors have a major role in shaping natural variation in body size, although the underlying mechanisms are poorly understood. Icelandic Arctic charr (Salvelinus alpinus L.) populations represent an ideal model to understand body‐size evolution, because adult dwarfism has arisen independently on multiple occasions in response to parallel environmental pressures. The mechanistic target of rapamycin (mTOR) pathway transmits signals from the environment to control cellular growth and is a primary candidate to be under selection for the dwarf phenotype. To test this hypothesis, we modified ‘inputs’ to this pathway in five dwarf and two generalist populations (with ancestral life history and body‐size traits), using a standardized manipulation of food intake in a common environment. The skeletal muscle transcript levels of 21 mTOR‐pathway genes were quantified in 274 individuals (∼6000 datapoints), and statistical modelling was used to elucidate sources of variation. Constitutive expression differences between populations were the main component of variation for around three‐quarters of the studied genes, irrespective of nutritional‐state and body‐size phenotype. There was evidence for stabilizing selection acting among populations, conserving the nutritionally dependent regulation of pathway genes controlling muscle atrophy. There were three genes (mTOR, 4E‐BP‐1 and IGFBP4), where the expression variation between dwarf and generalist populations exceeded the between‐population variation. Divergence in the expression of these candidate adaptive genes was most evident during a period of rapid growth following sustained fasting and was directionally consistent with their functions regulating growth and protein synthesis. We concluded that selection has operated efficiently to shape gene expression evolution in Icelandic charr populations and that the regulation of certain mTOR‐pathway genes evolved adaptively in locations favouring dwarfism, resulting in reduced muscle protein accretion under growth‐favouring conditions.

Development temperature has persistent effects on muscle growth responses in gilthead sea bream

Initially we characterised growth responses to altered nutritional input at the transcriptional and tissue levels in the fast skeletal muscle of juvenile gilthead sea bream. Fish reared at 21–22°C (range) were fed a commercial diet at 3% body mass d−1 (non-satiation feeding, NSF) for 4 weeks, fasted for 4d (F) and then fed to satiation (SF) for 21d. 13 out of 34 genes investigated showed consistent patterns of regulation between nutritional states. Fasting was associated with a 20-fold increase in MAFbx, and a 5-fold increase in Six1 and WASp expression, which returned to NSF levels within 16h of SF. Refeeding to satiation was associated with a rapid (<24 h) 12 to 17-fold increase in UNC45, Hsp70 and Hsp90α transcripts coding for molecular chaperones associated with unfolded protein response pathways. The growth factors FGF6 and IGF1 increased 6.0 and 4.5-fold within 16 h and 24 h of refeeding respectively. The average growth in diameter of fast muscle fibres was checked with fasting and significant fibre hypertrophy was only observed after 13d and 21d SF. To investigate developmental plasticity in growth responses we used the same experimental protocol with fish reared at either 17.5–18.5°C (range) (LT) or 21–22°C (range) (HT) to metamorphosis and then transferred to 21–22°C. There were persistent effects of development temperature on muscle growth patterns with 20% more fibres of lower average diameter in LT than HT group of similar body size. Altering the nutritional input to the muscle to stimulate growth revealed cryptic changes in the expression of UNC45 and Hsp90α with higher transcript abundance in the LT than HT groups, whereas there were no differences in the expression of MAFbx and Six1. It was concluded that myogenesis and gene expression patterns during growth are not fixed, but can be modified by temperature during the early stages of the life cycle.

Universal scaling rules predict evolutionary patterns of myogenesis in species with indeterminate growth

Intraspecific phenotypic variation is ubiquitous and often associated with resource exploitation in emerging habitats. For example, reduced body size has evolved repeatedly in Arctic charr (Salvelinus alpinus L.) and threespine stickleback (Gasterosteus aculeatus L.) across post-glacial habitats of the Northern Hemisphere. Exploiting these models, we examined how body size and myogenesis evolve with respect to the ‘optimum fibre size hypothesis’, which predicts that selection acts to minimize energetic costs associated with ionic homeostasis by optimizing muscle fibre production during development. In eight dwarf Icelandic Arctic charr populations, the ultimate production of fast-twitch muscle fibres (FNmax) was only 39.5 and 15.5 per cent of that in large-bodied natural and aquaculture populations, respectively. Consequently, average fibre diameter (FD) scaled with a mass exponent of 0.19, paralleling the relaxation of diffusional constraints associated with mass-specific metabolic rate scaling. Similar reductions in FNmax were observed for stickleback, including a small-bodied Alaskan population derived from a larger-bodied oceanic stock over a decadal timescale. The results suggest that in species showing indeterminate growth, body size evolution is accompanied by strong selection for fibre size optimization, theoretically allowing resources saved from ionic homeostasis to be allocated to other traits affecting fitness, including reproduction. Gene flow between small- and large-bodied populations residing in sympatry may counteract the evolution of this trait.

Comparison of the transcriptional responses of skeletal muscle and bone to a flooding dose of leucine in the gilthead sea bream (Sparus aurata)

Skeletal muscle, cartilage and bone must function in a co-ordinated fashion during locomotion and growth. In the present study on the gilthead sea bream (Sparus aurata) we tested the hypothesis that muscle and bone differ in their responsiveness to stimuli eliciting fast growth, providing a potential mechanism for generating the skeletal deformities observed in aquaculture. To investigate transcription regulation in skeletal muscle and bone we stimulated protein synthesis using a flooding dose of the branched chain amino acid leucine and compared the results with saline-injected controls. To increase the amount of available sequence information for gene expression analysis a de novo transcriptome was assembled using publicly available Next Generation Sequencing libraries from embryo, fast skeletal muscle, bone and cartilage. The resulting 5 million reads were assembled into 125,646 isotigs representing around 16,000 unique genes, including most components of the Pi3k/Akt/mTor signalling pathway. Principal components analysis was able to distinguish the transcriptional responses between leucine and saline injected controls in skeletal muscle, but not in the bone. General Linear Modelling revealed significant temporal changes in gene expression following leucine injection including the tissue-specific markers sparc, bglap (bone), mlc2 and myod2 (muscle) and gene transcripts associated with Pi3k/Akt/mTor signalling, p70sk6, akt2, ampka and mtor. Skeletal muscle showed more pronounced and rapid changes in transcript abundance than the bone to the same pro-growth signal. The observed differences in transcriptional response are consistent with the idea that fast growth results in a miss-match between muscle and bone development and may contribute to a higher incidence of skeletal deformities.