Temperature-associated selection linked to putative chromosomal inversions in king scallop (Pecten maximus).

The genomic landscape of divergence-the distribution of differences among populations or species across the genome-is increasingly characterized to understand the role that microevolutionary forces such as natural selection and recombination play in causing and maintaining genetic divergence. This line of inquiry has also revealed chromosome structure variation to be an important factor shaping the landscape of adaptive genetic variation. Owing to a high prevalence of chromosome structure variation and the strong pressure for local adaptation necessitated by their sessile nature, bivalve molluscs are an ideal taxon for exploring the relationship between chromosome structure variation and local adaptation. Here, we report a population genomic survey of king scallop (Pecten maximus) across its natural range in the northeastern Atlantic Ocean, using a recent chromosome-level genome assembly. We report the presence of at least three large (12-22 Mb), putative chromosomal inversions associated with sea surface temperature and whose frequencies are in contrast to neutral population structure. These results highlight a potentially large role for recombination-suppressing chromosomal inversions in local adaptation and suggest a hypothesis to explain the maintenance of differences in reproductive timing found at relatively small spatial scales across king scallop populations.

Influence of feed ration size on somatic and muscle growth in landlocked dwarf and farmed Atlantic salmon Salmo salar.

We examined the possible adaptation of the dwarf Bleke population of Atlantic salmon Salmo salar from Lake Byglandsfjord in southern Norway to limited food resources. The growth performance and muscle development in juvenile Bleke and farmed S. salar under satiated or restricted (50%) feeding were examined for 10 months, starting 3 weeks after first-feeding stage. Four-thousand fish were divided into four replicated groups and random samples of 16-40 fish per group were measured six times during the experiment. The two strains showed no significant difference in mean body mass when fed restricted ration, but the individual variation was considerably higher in the farmed fish. Both Bleke and farmed S. salar grew significantly faster when fed to satiation, but the farmed S. salar showed much higher gain in mass and were three times heavier (201.5 g vs 66.7 g) and possessed twice as many fast muscle fibres (179,682 vs 84,779) compared with landlocked S. salar after 10 months. Farmed fish fed full ration displayed both hypertrophic and hyperplasic muscle growth, while the increased growth in Bleke S. salar was entirely associated with a larger fibre diameter. The landlocked Bleke strain has apparently adapted to low food availability by minimising the metabolic costs of maintenance and growth through reduced dominance hierarchies and by an increase in average muscle fibre diameter relative to the ancestral condition.

Divergent regulation of insulin-like growth factor binding protein genes in cultured Atlantic salmon myotubes under different models of catabolism and anabolism.

Much attention has been given to insulin-like growth factor (Igf) pathways that regulate the balance of skeletal muscle protein synthesis and breakdown in response to a range of extrinsic and intrinsic signals. However, we have a less complete understanding of how the same signals modulate muscle mass upstream of such signalling, through a family of functionally-diverse Igf-binding proteins (Igfbps) that modify the availability of Igfs to the cell receptor Igf1r. We exposed cultured myotubes from Atlantic salmon (Salmo salar L.) to treatments recapturing three catabolic signals: inflammation (interleukin-1ß), stress (dexamethasone) and fasting (amino acid deprivation), plus one anabolic signal: recovery of muscle mass post-fasting (supplementation of fasted myotubes with Igf-I and amino acids). The intended phenotype of treatments was confirmed by significant changes in myotube diameter and immunofluorescent staining of structural proteins. We quantified the mRNA-level regulation of the full expressed Igf and Igfbp gene complement across a post-treatment time course, along with marker genes for muscle structural protein synthesis, as well as muscle breakdown, via the ubiquitin-proteasome and autophagy systems. Our results highlight complex, non-overlapping responses of Igfbp family members to the different treatments, suggesting that the profile of expressed Igfbps is differentially regulated by distinct signals promoting similar muscle remodelling phenotypes. We also demonstrate divergent regulation of salmonid-specific gene duplicates of igfbp5b1 and igfbp5b2 under distinct catabolic and anabolic conditions. Overall, this study increases our understanding of the regulation of Igfbp genes in response to signals that promote remodelling of skeletal muscle.

RNAseq analysis of fast skeletal muscle in restriction-fed transgenic coho salmon (Oncorhynchus kisutch): an experimental model uncoupling the growth hormone and nutritional signals regulating growth.

BACKGROUND: Coho salmon (Oncorhynchus kisutch) transgenic for growth hormone (Gh) express Gh in multiple tissues which results in increased appetite and continuous high growth with satiation feeding. Restricting Gh-transgenics to the same lower ration (TR) as wild-type fish (WT) results in similar growth, but with the recruitment of fewer, larger diameter, muscle skeletal fibres to reach a given body size. In order to better understand the genetic mechanisms behind these different patterns of muscle growth and to investigate how the decoupling of Gh and nutritional signals affects gene regulation we used RNA-seq to compare the fast skeletal muscle transcriptome in TR and WT coho salmon. RESULTS: Illumina sequencing of individually barcoded libraries from 6 WT and 6 TR coho salmon yielded 704,550,985 paired end reads which were used to construct 323,115 contigs containing 19,093 unique genes of which >10,000 contained >90 % of the coding sequence. Transcripts coding for 31 genes required for myoblast fusion were identified with 22 significantly downregulated in TR relative to WT fish, including 10 (vaspa, cdh15, graf1, crk, crkl, dock1, trio, plekho1a, cdc42a and dock5) associated with signaling through the cell surface protein cadherin. Nineteen out of 44 (43 %) translation initiation factors and 14 of 47 (30 %) protein chaperones were upregulated in TR relative to WT fish. CONCLUSIONS: TR coho salmon showed increased growth hormone transcripts and gene expression associated with protein synthesis and folding than WT fish even though net rates of protein accretion were similar. The uncoupling of Gh and amino acid signals likely results in additional costs of transcription associated with protein turnover in TR fish. The predicted reduction in the ionic costs of homeostasis in TR fish associated with increased fibre size were shown to involve multiple pathways regulating myotube fusion, particularly cadherin signaling.

Characterization of the transcriptome of fast and slow muscle myotomal fibres in the pacu (Piaractus mesopotamicus).

BACKGROUND: The Pacu (Piaractus mesopotamicus) is a member of the Characiform family native to the Prata Basin (South America) and a target for the aquaculture industry. A limitation for the development of a selective breeding program for this species is a lack of available genetic information. The primary objectives of the present study were 1) to increase the genetic resources available for the species, 2) to exploit the anatomical separation of myotomal fibres types to compare the transcriptomes of slow and fast muscle phenotypes and 3) to systematically investigate the expression of Ubiquitin Specific Protease (USP) family members in fast and slow muscle in response to fasting and refeeding. RESULTS: We generated 0.6 Tb of pair-end reads from slow and fast skeletal muscle libraries. Over 665 million reads were assembled into 504,065 contigs with an average length of 1,334 bp and N50 = 2,772 bp. We successfully annotated nearly 47% of the transcriptome and identified around 15,000 unique genes and over 8000 complete coding sequences. 319 KEGG metabolic pathways were also annotated and 380 putative microsatellites were identified. 956 and 604 genes were differentially expressed between slow and fast skeletal muscle, respectively. 442 paralogues pairs arising from the teleost-specific whole genome duplication were identified, with the majority showing different expression patterns between fibres types (301 in slow and 245 in fast skeletal muscle). 45 members of the USP family were identified in the transcriptome. Transcript levels were quantified by qPCR in a separate fasting and refeeding experiment. USP genes in fast muscle showed a similar transient increase in expression with fasting as the better characterized E3 ubiquitin ligases. CONCLUSION: We have generated a 53-fold coverage transcriptome for fast and slow myotomal muscle in the pacu (Piaractus mesopotamicus) significantly increasing the genetic resources available for this important aquaculture species. We describe significant differences in gene expression between muscle fibre types for fundamental components of general metabolism, the Pi3k/Akt/mTor network and myogenesis, including detailed analysis of paralogue expression. We also provide a comprehensive description of USP family member expression between muscle fibre types and with changing nutritional status.

Temperature during embryonic development has persistent effects on thermal acclimation capacity in zebrafish.

Global warming is intensifying interest in the mechanisms enabling ectothermic animals to adjust physiological performance and cope with temperature change. Here we show that embryonic temperature can have dramatic and persistent effects on thermal acclimation capacity at multiple levels of biological organization. Zebrafish embryos were incubated until hatching at control temperature (T(E) = 27 °C) or near the extremes for normal development (T(E) = 22 °C or 32 °C) and were then raised to adulthood under common conditions at 27 °C. Short-term temperature challenge affected aerobic exercise performance (U(crit)), but each T(E) group had reduced thermal sensitivity at its respective T(E). In contrast, unexpected differences arose after long-term acclimation to 16 °C, when performance in the cold was ∼20% higher in both 32 °C and 22 °C T(E) groups compared with 27 °C T(E) controls. Differences in performance after acclimation to cold or warm (34 °C) temperatures were partially explained by variation in fiber type composition in the swimming muscle. Cold acclimation changed the abundance of 3,452 of 19,712 unique and unambiguously identified transcripts detected in the fast muscle using RNA-Seq. Principal components analysis differentiated the general transcriptional responses to cold of the 27 °C and 32 °C T(E) groups. Differences in expression were observed for individual genes involved in energy metabolism, angiogenesis, cell stress, muscle contraction and remodeling, and apoptosis. Therefore, thermal acclimation capacity is not fixed and can be modified by temperature during early development. Developmental plasticity may thus help some ectothermic organisms cope with the more variable temperatures that are expected under future climate-change scenarios.

Profiling of the embryonic Atlantic halibut (Hippoglossus hippoglossus L.) transcriptome reveals maternal transcripts as potential markers of embryo quality.

BACKGROUND: Commercial Atlantic halibut (Hippoglossus hippoglossus) farming is restricted by variable oocyte quality, slow growth, and early maturation of male fish. Maternally transferred components regulate early developmental processes; therefore, they have an effect on the future viability of the embryo. Using a newly developed Agilent 10 k custom-made oligonucleotide array, we profiled components of the transcriptome involved in immune defence as well as germline and muscle development during early developmental stages: 8-cell embryos (8CS), germ ring stage (GR), 10-somite stage (10SS), and hatched embryos (HT). In addition, we identified differentially expressed transcripts in low (≤9 ± 3% hatching) and high (≥86 ± 3°% hatching) quality eggs at 8CS to identify potential maternal markers for embryo quality. RESULTS: Out of 2066 differentially expressed transcripts, 160 were identified as maternal transcripts being specifically expressed at 8CS only. Twenty transcripts were differentially expressed in 8-cell embryos between low and high quality egg groups. Several immune-related transcripts were identified as promising molecular markers of hatching success including interferon regulatory factor 7 and mhc class 2A chain. Differential expression was positively validated with quantitative real-time PCR. CONCLUSIONS: We have demonstrated maternal transfer of innate and adaptive immune system transcripts into Atlantic halibut embryos and their relation with future embryo developmental potential. We identified several transcripts as potential molecular markers of embryo quality. The developed microarray represents a useful resource for improving the commercial production of Atlantic halibut.

Evolution of ancient functions in the vertebrate insulin-like growth factor system uncovered by study of duplicated salmonid fish genomes.

Whole-genome duplication (WGD) was experienced twice by the vertebrate ancestor (2 rounds; 2R), again by the teleost fish ancestor (3R) and most recently in certain teleost lineages (4R). Consequently, vertebrate gene families are often expanded in 3R and 4R genomes. Arguably, many types of "functional divergence" present across 2R gene families will exceed that between 3R/4R paralogs of genes comprising 2R families. Accordingly, 4R offers a form of replication of 2R. Examining whether this concept has implications for molecular evolutionary research, we studied insulin-like growth factor (IGF) binding proteins (IGFBPs), whose six 2R family members carry IGF hormones and regulate interactions between IGFs and IGF1-receptors (IGF1Rs). Using phylogenomic approaches, we resolved the complete IGFBP repertoire of 4R-derived salmonid fishes (19 genes; 13 more than human) and established evolutionary relationships/nomenclature with respect to WGDs. Traits central to IGFBP action were determined for all genes, including atomic interactions in IGFBP-IGF1/IGF2 complexes regulating IGF-IGF1R binding. Using statistical methods, we demonstrate that attributes of these protein interfaces are overwhelming a product of 2R IGFBP family membership, explain 49-68% of variation in IGFBP mRNA concentration in several different tissues, and strongly predict the strength and direction of IGFBP transcriptional regulation under differing nutritional states. The results support a model where vertebrate IGFBP family members evolved divergent structural attributes to provide distinct competition for IGFs with IGF1Rs, predisposing different functions in the regulation of IGF signaling. Evolution of gene expression then acted to ensure the appropriate physiological production of IGFBPs according to their structural specializations, leading to optimal IGF-signaling according to nutritional-status and the endocrine/local mode of action. This study demonstrates that relatively recent gene family expansion can facilitate inference of functional evolution within ancient genetic systems.

A well-constrained estimate for the timing of the salmonid whole genome duplication reveals major decoupling from species diversification.

Whole genome duplication (WGD) is often considered to be mechanistically associated with species diversification. Such ideas have been anecdotally attached to a WGD at the stem of the salmonid fish family, but remain untested. Here, we characterized an extensive set of gene paralogues retained from the salmonid WGD, in species covering the major lineages (subfamilies Salmoninae, Thymallinae and Coregoninae). By combining the data in calibrated relaxed molecular clock analyses, we provide the first well-constrained and direct estimate for the timing of the salmonid WGD. Our results suggest that the event occurred no later in time than 88 Ma and that 40-50 Myr passed subsequently until the subfamilies diverged. We also recovered a Thymallinae-Coregoninae sister relationship with maximal support. Comparative phylogenetic tests demonstrated that salmonid diversification patterns are closely allied in time with the continuous climatic cooling that followed the Eocene-Oligocene transition, with the highest diversification rates coinciding with recent ice ages. Further tests revealed considerably higher speciation rates in lineages that evolved anadromy--the physiological capacity to migrate between fresh and seawater--than in sister groups that retained the ancestral state of freshwater residency. Anadromy, which probably evolved in response to climatic cooling, is an established catalyst of genetic isolation, particularly during environmental perturbations (for example, glaciation cycles). We thus conclude that climate-linked ecophysiological factors, rather than WGD, were the primary drivers of salmonid diversification.

Muscle fibre size optimisation provides flexibility for energy budgeting in calorie-restricted coho salmon transgenic for growth hormone.

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.

Cardiac myoglobin deficit has evolved repeatedly in teleost fishes.

Myoglobin (Mb) is the classic vertebrate oxygen-binding protein present in aerobic striated muscles. It functions principally in oxygen delivery and provides muscle with its characteristic red colour. Members of the Antarctic icefish family (Channichthyidae) are widely thought to be extraordinary for lacking cardiac Mb expression, a fact that has been attributed to their low metabolic rate and unusual evolutionary history. Here, we report that cardiac Mb deficit, associated with pale heart colour, has evolved repeatedly during teleost evolution. This trait affects both gill- and air-breathing species from temperate to tropical habitats across a full range of salinities. Cardiac Mb deficit results from total pseudogenization in three-spined stickleback and is associated with a massive reduction in mRNA level in two species that evidently retain functional Mb. The results suggest that near or complete absence of Mb-assisted oxygen delivery to heart muscle is a common facet of teleost biodiversity, even affecting lineages with notable oxygen demands. We suggest that Mb deficit may affect how different teleost species deal with increased tissue oxygen demands arising under climate change.

Characterisation and expression of myogenesis regulatory factors during in vitro myoblast development and in vivo fasting in the gilthead sea bream (Sparus aurata).

The aim of this study was to characterise a primary cell culture isolated from fast skeletal muscle of the gilthead sea bream. Gene expression profiles during culture maturation were compared with those obtained from a fasting-refeeding model which is widely used to modulate myogenesis in vivo. Myogenesis is controlled by numerous extracellular signals together with intracellular transcriptional factors whose coordinated expression is critical for the appropriate development of muscle fibres. Full-length cDNAs for the transcription factors Myf5, Mrf4, Pax7 and Sox8 were cloned and sequenced for gilthead sea bream. Pax7, sox8, myod2 and myf5 levels were up-regulated during the proliferating phase of the myogenic cultures coincident with the highest expression of proliferating cell nuclear antigen (PCNA). In contrast, myogenin and mrf4 transcript abundance was highest during the differentiation phase of the culture when myotubes were present, and was correlated with increased myosin heavy chain (mhc) and desmin expression. In vivo, 30days of fasting resulted in muscle fibre atrophy, a reduction in myod2, myf5 and igf1 expression, lower number of Myod-positive cells, and decreased PCNA protein expression, whereas myogenin expression was not significantly affected. Myostatin1 (mstn1) and pax7 expression were up-regulated in fasted relative to well-fed individuals, consistent with a role for Pax7 in the reduction of myogenic cell activity with fasting. The primary cell cultures and fasting-feeding experiments described provide a foundation for the future investigations on the regulation of muscle growth in gilthead sea bream.

Stac3 is required for myotube formation and myogenic differentiation in vertebrate skeletal muscle.

Stac3 was identified as a nutritionally regulated gene from an Atlantic salmon subtractive hybridization library with highest expression in skeletal muscle. Salmon Stac3 mRNA was highly correlated with myogenin and myoD1a expression during differentiation of a salmon primary myogenic culture and was regulated by amino acid availability. In zebrafish embryos, stac3 was initially expressed in myotomal adaxial cells and in fast muscle fibers post-segmentation. Morpholino knockdown resulted in defects in myofibrillar protein assembly, particularly in slow muscle fibers, and decreased levels of the hedgehog receptor patched. The function of Stac3 was further characterized in vitro using the mammalian C2C12 myogenic cell line. Stac3 mRNA expression increased during the differentiation of the C2C12 myogenic cell line. Knockdown of Stac3 by RNAi inhibited myotube formation, and microarray analysis revealed that transcripts involved in cell cycle, focal adhesion, cytoskeleton, and the pro-myogenic factors Igfbp-5 and Igf2 were down-regulated. RNAi-treated cells had suppressed Akt signaling and exogenous insulin-like growth factor (Igf) 2 was unable to rescue the phenotype, however, Igf/Akt signaling was not blocked. Overexpression of Stac3, which results in increased levels of Igfbp-5 mRNA, did not lead to increased differentiation. In synchronized cells, Stac3 mRNA was most abundant during the G(1) phase of the cell cycle. RNAi-treated cells were smaller, had higher proliferation rates and a decreased proportion of cells in G(1) phase when compared with controls, suggesting a role in the G(1) phase checkpoint. These results identify Stac3 as a new gene required for myogenic differentiation and myofibrillar protein assembly in vertebrates.

Evaluation of low-density SNP panels and imputation for cost-effective genomic selection in four aquaculture species.

Genomic selection can accelerate genetic progress in aquaculture breeding programmes, particularly for traits measured on siblings of selection candidates. However, it is not widely implemented in most aquaculture species, and remains expensive due to high genotyping costs. Genotype imputation is a promising strategy that can reduce genotyping costs and facilitate the broader uptake of genomic selection in aquaculture breeding programmes. Genotype imputation can predict ungenotyped SNPs in populations genotyped at a low-density (LD), using a reference population genotyped at a high-density (HD). In this study, we used datasets of four aquaculture species (Atlantic salmon, turbot, common carp and Pacific oyster), phenotyped for different traits, to investigate the efficacy of genotype imputation for cost-effective genomic selection. The four datasets had been genotyped at HD, and eight LD panels (300-6,000 SNPs) were generated in silico. SNPs were selected to be: i) evenly distributed according to physical position ii) selected to minimise the linkage disequilibrium between adjacent SNPs or iii) randomly selected. Imputation was performed with three different software packages (AlphaImpute2, FImpute v.3 and findhap v.4). The results revealed that FImpute v.3 was faster and achieved higher imputation accuracies. Imputation accuracy increased with increasing panel density for both SNP selection methods, reaching correlations greater than 0.95 in the three fish species and 0.80 in Pacific oyster. In terms of genomic prediction accuracy, the LD and the imputed panels performed similarly, reaching values very close to the HD panels, except in the pacific oyster dataset, where the LD panel performed better than the imputed panel. In the fish species, when LD panels were used for genomic prediction without imputation, selection of markers based on either physical or genetic distance (instead of randomly) resulted in a high prediction accuracy, whereas imputation achieved near maximal prediction accuracy independently of the LD panel, showing higher reliability. Our results suggests that, in fish species, well-selected LD panels may achieve near maximal genomic selection prediction accuracy, and that the addition of imputation will result in maximal accuracy independently of the LD panel. These strategies represent effective and affordable methods to incorporate genomic selection into most aquaculture settings.

Fast skeletal muscle transcriptome of the gilthead sea bream (Sparus aurata) determined by next generation sequencing.

BACKGROUND: The gilthead sea bream (Sparus aurata L.) occurs around the Mediterranean and along Eastern Atlantic coasts from Great Britain to Senegal. It is tolerant of a wide range of temperatures and salinities and is often found in brackish coastal lagoons and estuarine areas, particularly early in its life cycle. Gilthead sea bream are extensively cultivated in the Mediterranean with an annual production of 125,000 metric tonnes. Here we present a de novo assembly of the fast skeletal muscle transcriptome of gilthead sea bream using 454 reads and identify gene paralogues, splice variants and microsatellite repeats. An annotated transcriptome of the skeletal muscle will facilitate understanding of the genetic and molecular basis of traits linked to production in this economically important species. RESULTS: Around 2.7 million reads of mRNA sequence data were generated from the fast myotomal of adult fish (~2 kg) and juvenile fish (~0.09 kg) that had been either fed to satiation, fasted for 3-5d or transferred to low (11°C) or high (33°C) temperatures for 3-5d. Newbler v2.5 assembly resulted in 43,461 isotigs >100 bp. The number of sequences annotated by searching protein and gene ontology databases was 10,465. The average coverage of the annotated isotigs was x40 containing 5655 unique gene IDs and 785 full-length cDNAs coding for proteins containing 58-1536 amino acids. The v2.5 assembly was found to be of good quality based on validation using 200 full-length cDNAs from GenBank. Annotated isotigs from the reference transcriptome were attributable to 344 KEGG pathway maps. We identified 26 gene paralogues (20 of them teleost-specific) and 43 splice variants, of which 12 had functional domains missing that were likely to affect their biological function. Many key transcription factors, signaling molecules and structural proteins necessary for myogenesis and muscle growth have been identified. Physiological status affected the number of reads that mapped to isotigs, reflecting changes in gene expression between treatments. CONCLUSIONS: We have produced a comprehensive fast skeletal muscle transcriptome for the gilthead sea bream, which will provide a resource for SNP discovery in genes with a large effect on production traits of commercial interest and for expression studies of growth and adaptation.

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 (FN(max)) 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 FN(max) 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.

Circadian expression of clock and putative clock-controlled genes in skeletal muscle of the zebrafish.

To identify circadian patterns of gene expression in skeletal muscle, adult male zebrafish were acclimated for 2 wk to a 12:12-h light-dark photoperiod and then exposed to continuous darkness for 86 h with ad libitum feeding. The increase in gut food content associated with the subjective light period was much diminished by the third cycle, enabling feeding and circadian rhythms to be distinguished. Expression of zebrafish paralogs of mammalian transcriptional activators of the circadian mechanism (bmal1, clock1, and rora) followed a rhythmic pattern with a ∼24-h periodicity. Peak expression of rora paralogs occurred at the beginning of the subjective light period [Zeitgeber time (ZT)07 and ZT02 for roraa and rorab], whereas the highest expression of bmal1 and clock paralogs occurred 12 h later (ZT13-15 and ZT16 for bmal and clock paralogs). Expression of the transcriptional repressors cry1a, per1a/1b, per2, per3, nr1d2a/2b, and nr1d1 also followed a circadian pattern with peak expression at ZT0-02. Expression of the two paralogs of cry2 occurred in phase with clock1a/1b. Duplicated genes had a high correlation of expression except for paralogs of clock1, nr1d2, and per1, with cry1b showing no circadian pattern. The highest expression difference was 9.2-fold for the activator bmal1b and 51.7-fold for the repressor per1a. Out of 32 candidate clock-controlled genes, only myf6, igfbp3, igfbp5b, and hsf2 showed circadian expression patterns. Igfbp3, igfbp5b, and myf6 were expressed in phase with clock1a/1b and had an average of twofold change in expression from peak to trough, whereas hsf2 transcripts were expressed in phase with cry1a and had a 7.2-fold-change in expression. The changes in expression of clock and clock-controlled genes observed during continuous darkness were also observed at similar ZTs in fish exposed to a normal photoperiod in a separate control experiment. The role of circadian clocks in regulating muscle maintenance and growth are discussed.

Experimental selection for body size at age modifies early life-history traits and muscle gene expression in adult zebrafish.

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.

Postprandial expression of growth-related genes in Atlantic salmon (Salmo salar L.) juveniles fasted for 1 week and fed a single meal to satiation.

We investigated postprandial changes in transcript abundance following a single satiating meal in juvenile Atlantic salmon (Salmo salar L.) (about 70 g body mass) following fasting for 1 week at 12°C. The expression of twenty-three growth-related genes was determined in fast myotomal muscle using quantitative real-time PCR at the following postprandial time points: - 12, 0, 1, 3, 6, 12, 24, 48 and 96 h. The gut was fullest 1-6 h after feeding and emptied within 48-96 h. IGF-I, MyoD1c, MRF4 and myf5 transcripts were sharply up-regulated within 1 h of refeeding and are promising candidate genes involved in a fast-response signalling system that regulates fish myotomal muscle growth. These genes clustered together with MyoD1b and suggest a coordinated regulation to favour resumption of myogenesis as an early response to feeding. Insulin-like growth factor (IGF)-II and the ubiquitin ligase MAFbx/atrogin-1 were initially down-regulated but restored to initial values after 12 h. It is also suggested that local production of IGF-I within the muscle might suppress catabolic pathways depressing MAFbx/atrogin-1.

Targeted rapid amplification of cDNA ends (T-RACE)--an improved RACE reaction through degradation of non-target sequences.

Amplification of the 5' ends of cDNA, although simple in theory, can often be difficult to achieve. We describe a novel method for the specific amplification of cDNA ends. An oligo-dT adapter incorporating a dUTP-containing PCR primer primes first-strand cDNA synthesis incorporating dUTP. Using the Cap finder approach, another distinct dUTP containing adapter is added to the 3' end of the newly synthesized cDNA. Second-strand synthesis incorporating dUTP is achieved by PCR, using dUTP-containing primers complimentary to the adapter sequences incorporated in the cDNA ends. The double-stranded cDNA-containing dUTP serves as a universal template for the specific amplification of the 3' or 5' end of any gene. To amplify the ends of cDNA, asymmetric PCR is performed using a single gene-specific primer and standard dNTPs. The asymmetric PCR product is purified and non-target transcripts containing dUTP degraded by Uracil DNA glycosylase, leaving only those transcripts produced during the asymmetric PCR. Subsequent PCR using a nested gene-specific primer and the 3' or 5' T-RACE primer results in specific amplification of cDNA ends. This method can be used to specifically amplify the 3' and 5' ends of numerous cDNAs from a single cDNA synthesis reaction.