Exploring the Integrated Role of miRNAs and lncRNAs in Regulating the Transcriptional Response to Amino Acids and Insulin-like Growth Factor 1 in Gilthead Sea Bream (Sparus aurata) Myoblasts.

In this study, gilthead sea bream (Sparus aurata) fast muscle myoblasts were stimulated with two pro-growth treatments, amino acids (AA) and insulin-like growth factor 1 (Igf-1), to analyze the transcriptional response of mRNAs, microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) and to explore their possible regulatory network using bioinformatic approaches. AA had a higher impact on transcription (1795 mRNAs changed) compared to Igf-1 (385 mRNAs changed). Both treatments stimulated the transcription of mRNAs related to muscle differentiation (GO:0042692) and sarcomere (GO:0030017), while AA strongly stimulated DNA replication and cell division (GO:0007049). Both pro-growth treatments altered the transcription of over 100 miRNAs, including muscle-specific miRNAs (myomiRs), such as miR-133a/b, miR-206, miR-499, miR-1, and miR-27a. Among 111 detected lncRNAs (>1 FPKM), only 30 were significantly changed by AA and 11 by Igf-1. Eight lncRNAs exhibited strong negative correlations with several mRNAs, suggesting a possible regulation, while 30 lncRNAs showed strong correlations and interactions with several miRNAs, suggesting a role as sponges. This work is the first step in the identification of the ncRNAs network controlling muscle development and growth in gilthead sea bream, pointing out potential regulatory mechanisms in response to pro-growth signals.

Improving the Aerobic Capacity in Fingerlings of European Sea Bass (Dicentrarchus labrax) through Moderate and Sustained Exercise: A Metabolic Approach.

Sustained swimming induces beneficial effects on growth and energy metabolism in some fish species. However, the absence of a standardized exercise regimen that guarantees an optimal response to physical activity is due to the anatomical, behavioral, and physiological differences among species, and the different conditions of tests applied, which are especially notable for the early stages of cultured species. The objective of this study was to assess the growth and metabolic responses of European sea bass submitted to continuous and moderate exercise exposure, selecting a practical swimming speed from swimming tests of groups of five fingerlings. The exercise-effects trial was carried out with 600 sea bass fingerlings (3-5 g body weight) distributed in two groups (control: voluntary swimming; exercised: under sustained swimming at 1.5 body lengths·s-1). After 6 weeks, growth parameters and proximal composition of both muscles were not altered by sustained swimming, but an increased synthetic capacity (increased RNA/DNA ratio) and more efficient use of proteins (decreased ΔN15) were observed in white muscle. The gene expression of mitochondrial proteins in white and red muscle was not affected by exercise, except for ucp3, which increased. The increase of UCP3 and Cox4 protein expression, as well as the higher COX/CS ratio of enzyme activity in white muscle, pointed out an enhanced oxidative capacity in this tissue during sustained swimming. In the protein expression of red muscle, only CS increased. All these metabolic adaptations to sustained exercise were also reflected in an enhanced maximum metabolic rate (MMR) with higher aerobic scope (AMS) of exercised fish in comparison to the non-trained fish, during a swimming test. These results demonstrated that moderate sustained swimming applied to sea bass fingerlings can improve the physical fitness of individuals through the enhancement of their aerobic capacities.

Muscle regeneration in gilthead sea bream: Implications of endocrine and local regulatory factors and the crosstalk with bone.

Fish muscle regeneration is still a poorly known process. In the present study, an injury was done into the left anterior epaxial skeletal muscle of seventy 15 g gilthead sea bream (Sparus aurata) juveniles to evaluate at days 0, 1, 2, 4, 8, 16 and 30 post-wound, the expression of several muscle genes. Moreover, transcripts' expression in the bone (uninjured tissue) was also analyzed. Histology of the muscle showed the presence of dead tissue the first day after injury and how the damaged fibers were removed and replaced by new muscle fibers by day 16 that kept growing up to day 30. Gene expression results showed in muscle an early upregulation of igf-2 and a downregulation of ghr-1 and igf-1. Proteolytic systems expression increased with capn2 and ctsl peaking at 1 and 2 days post-injury, respectively and mafbx at day 8. A pattern of expression that fitted well with active myogenesis progression 16 days after the injury was then observed, with the recovery of igf-1, pax7, cmet, and cav1 expression; and later on, that of cav3 as well. Furthermore, the first days post-injury, the cytokines il-6 and il-15 were also upregulated confirming the tissue inflammation, while tnfα was only upregulated at days 16 and 30 to induce satellite cells recruitment; overall suggesting a possible role for these molecules as myokines. The results of the bone transcripts showed an upregulation first, of bmp2 and ctsk at days 1 and 2, respectively; then, ogn1 and ocn peaked at day 4 in parallel to mstn2 downregulation, and runx2 and ogn2 increased after 8 days of muscle injury, suggesting a possible tissue crosstalk during the regenerative process. Overall, the present model allows studying the sequential involvement of different regulatory molecules during muscle regeneration, as well as the potential relationship between muscle and other tissues such as bone to control musculoskeletal development and growth, pointing out an interesting new line of research in this group of vertebrates.

Myomaker and Myomixer Characterization in Gilthead Sea Bream under Different Myogenesis Conditions.

Skeletal muscle is formed by multinucleated myofibers originated by waves of hyperplasia and hypertrophy during myogenesis. Tissue damage triggers a regeneration process including new myogenesis and muscular remodeling. During myogenesis, the fusion of myoblasts is a key step that requires different genes' expression, including the fusogens myomaker and myomixer. The present work aimed to characterize these proteins in gilthead sea bream and their possible role in in vitro myogenesis, at different fish ages and during muscle regeneration after induced tissue injury. Myomaker is a transmembrane protein highly conserved among vertebrates, whereas Myomixer is a micropeptide that is moderately conserved. myomaker expression is restricted to skeletal muscle, while the expression of myomixer is more ubiquitous. In primary myocytes culture, myomaker and myomixer expression peaked at day 6 and day 8, respectively. During regeneration, the expression of both fusogens and all the myogenic regulatory factors showed a peak after 16 days post-injury. Moreover, myomaker and myomixer were present at different ages, but in fingerlings there were significantly higher transcript levels than in juveniles or adult fish. Overall, Myomaker and Myomixer are valuable markers of muscle growth that together with other regulatory molecules can provide a deeper understanding of myogenesis regulation in fish.

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.

Fast-growing growth hormone transgenic coho salmon (Oncorhynchus kisutch) show a lower incidence of vaterite deposition and malformations in sagittal otoliths.

In fish otoliths, CaCO3 normally precipitates as aragonite, and more rarely as vaterite or calcite. A higher incidence of vaterite deposition in otoliths from aquaculture-reared fish has been reported and it is thought that high growth rates under farming conditions might promote its deposition. To test this hypothesis, otoliths from growth hormone (GH) transgenic coho salmon and non-transgenic fish of matching size were compared. Once morphometric parameters were normalized by animal length, we found that transgenic fish otoliths were smaller (-24%, -19%, -20% and -30% for length, width, perimeter and area, respectively; P<0.001) and rounder (-12%, +13.5%, +15% and -15.5% in circularity, form factor, roundness and ellipticity; P<0.001) than otoliths from non-transgenic fish of matching size. Interestingly, transgenic fish had smaller eyes (-30% eye diameter) and showed a strong correlation between eye and otolith size. We also found that the percentage of otoliths showing vaterite deposition was significantly smaller in transgenic fish (21-28%) than in non-transgenic fish (69%; P<0.001). Likewise, the area affected by vaterite deposition within individual otoliths was reduced in transgenic fish (21-26%) compared with non-transgenic fish (42.5%; P<0.001). Our results suggest that high growth rates per se are not sufficient to cause vaterite deposition in all cases, and that GH overexpression might have a protective role against vaterite deposition, a hypothesis that needs further investigation.

The Emerging Role of Long Non-Coding RNAs in Development and Function of Gilthead Sea Bream (Sparus aurata) Fast Skeletal Muscle.

Long non-coding RNAs (lncRNAs) are an emerging group of ncRNAs that can modulate gene expression at the transcriptional or translational levels. In the present work, previously published transcriptomic data were used to identify lncRNAs expressed in gilthead sea bream skeletal muscle, and their transcription levels were studied under different physiological conditions. Two hundred and ninety lncRNAs were identified and, based on transcriptomic differences between juveniles and adults, a total of seven lncRNAs showed potential to be important for muscle development. Our data suggest that the downregulation of most of the studied lncRNAs might be linked to increased myoblast proliferation, while their upregulation might be necessary for differentiation. However, with these data, as it is not possible to propose a formal mechanism to explain their effect, bioinformatic analysis suggests two possible mechanisms. First, the lncRNAs may act as sponges of myoblast proliferation inducers microRNAs (miRNAs) such as miR-206, miR-208, and miR-133 (binding energy MEF < -25.0 kcal). Secondly, lncRNA20194 had a strong predicted interaction towards the myod1 mRNA (ndG = -0.17) that, based on the positive correlation between the two genes, might promote its function. Our study represents the first characterization of lncRNAs in gilthead sea bream fast skeletal muscle and provides evidence regarding their involvement in muscle development.

Amino Acids and IGF1 Regulation of Fish Muscle Growth Revealed by Transcriptome and microRNAome Integrative Analyses of Pacu (Piaractus mesopotamicus) Myotubes.

Amino acids (AA) and IGF1 have been demonstrated to play essential roles in protein synthesis and fish muscle growth. The myoblast cell culture is useful for studying muscle regulation, and omics data have contributed enormously to understanding its molecular biology. However, to our knowledge, no study has performed the large-scale sequencing of fish-cultured muscle cells stimulated with pro-growth signals. In this work, we obtained the transcriptome and microRNAome of pacu (Piaractus mesopotamicus)-cultured myotubes treated with AA or IGF1. We identified 1228 and 534 genes differentially expressed by AA and IGF1. An enrichment analysis showed that AA treatment induced chromosomal changes, mitosis, and muscle differentiation, while IGF1 modulated IGF/PI3K signaling, metabolic alteration, and matrix structure. In addition, potential molecular markers were similarly modulated by both treatments. Muscle-miRNAs (miR-1, -133, -206 and -499) were up-regulated, especially in AA samples, and we identified molecular networks with omics integration. Two pairs of genes and miRNAs demonstrated a high-level relationship, and involvement in myogenesis and muscle growth: marcksb and miR-29b in AA, and mmp14b and miR-338-5p in IGF1. Our work helps to elucidate fish muscle physiology and metabolism, highlights potential molecular markers, and creates a perspective for improvements in aquaculture and in in vitro meat production.

The Effect of 17α-Ethynilestradiol and GPER1 Activation on Body and Muscle Growth, Muscle Composition and Growth-Related Gene Expression of Gilthead Seabream, Sparus aurata L.

Endocrine-disrupting chemicals include natural and synthetic estrogens, such as 17α-ethynilestradiol (EE2), which can affect reproduction, growth and immunity. Estrogen signalling is mediated by nuclear or membrane estrogen receptors, such as the new G-protein-coupled estrogen receptor 1 (GPER1). The present work studies the effect of EE2 and G1 (an agonist of GPER1) on body and muscle parameters and growth-related genes of 54 two-year-old seabreams. The fish were fed a diet containing EE2 (EE2 group) and G1 (G1 group) for 45 days and then a diet without EE2 or G1 for 122 days. An untreated control group was also studied. At 45 days, the shortest body length was observed in the G1 group, while 79 and 122 days after the cessation of treatments, the shortest body growth was observed in the EE2 group. Hypertrophy of white fibers was higher in the EE2 and G1 groups than it was in the control group, whereas the opposite was the case with respect to hyperplasia. Textural hardness showed a negative correlation with the size of white fibers. At the end of the experiment, all fish analyzed in the EE2 group showed a predominance of the gonadal ovarian area. In addition, the highest expression of the mafbx gene (upregulated in catabolic signals) and mstn2 (myogenesis negative regulator) was found in EE2-exposed fish.

Pilot study to investigate the effect of long-term exposure to high pCO2 on adult cod (Gadus morhua) otolith morphology and calcium carbonate deposition.

To date the study of ocean acidification on fish otolith formation has been mainly focused on larval and juvenile stages. In the present pilot study, wild-captured adult Atlantic cod (Gadus morhua) were exposed to two different levels of pCO2, 422µatm (ambient, low pCO2) or 1091µatm (high pCO2), for a period of 30 weeks (from mid-October to early April 2014-2015) in order to study the effects on otolith size, shape and CaCO3 crystallization amongst other biological parameters. We found that otoliths from cod exposed to high pCO2 were slightly smaller (- 3.4% in length; - 3.3% in perimeter), rounder (- 2.9% circularity and + 4% roundness) but heavier (+ 5%) than the low pCO2 group. Interestingly, there were different effects in males and females; for instance, male cods exposed to high pCO2 exhibited significant changes in circularity (- 3%) and roundness (+ 4%) compared to the low pCO2 males, but without significant changes on otolith dimensions, while females exposed to high pCO2 had smaller otoliths as shown for length (- 5.6%), width (- 2%), perimeter (- 3.5%) and area (- 4.8%). Furthermore, while the majority of the otoliths analysed showed normal aragonite deposition, 10% of fish exposed to 1091µatm of pCO2 had an abnormal accretion of calcite, suggesting a shift on calcium carbonate polymorph crystallization in some individuals under high pCO2 conditions. Our preliminary results indicate that high levels of pCO2 in adult Atlantic cod might affect otolith growth in a gender-specific way. Our findings reveal that otoliths from adult cod are affected by ocean acidification, and we believe that the present study will prompt further research into this currently under-explored area.

An insight on the impact of teleost whole genome duplication on the regulation of the molecular networks controlling skeletal muscle growth.

Fish muscle growth is a complex process regulated by multiple pathways, resulting on the net accumulation of proteins and the activation of myogenic progenitor cells. Around 350-320 million years ago, teleost fish went through a specific whole genome duplication (WGD) that expanded the existent gene repertoire. Duplicated genes can be retained by different molecular mechanisms such as subfunctionalization, neofunctionalization or redundancy, each one with different functional implications. While the great majority of ohnolog genes have been identified in the teleost genomes, the effect of gene duplication in the fish physiology is still not well characterized. In the present study we studied the effect of WGD on the transcription of the duplicated components controlling muscle growth. We compared the expression of lineage-specific ohnologs related to myogenesis and protein balance in the fast-skeletal muscle of pacus (Piaractus mesopotamicus-Ostariophysi) and Nile tilapias (Oreochromis niloticus-Acanthopterygii) fasted for 4 days and refed for 3 days. We studied the expression of 20 ohnologs and found that in the great majority of cases, duplicated genes had similar expression profiles in response to fasting and refeeding, indicating that their functions during growth have been conserved during the period after the WGD. Our results suggest that redundancy might play a more important role in the retention of ohnologs of regulatory pathways than initially thought. Also, comparison to non-duplicated orthologs showed that it might not be uncommon for the duplicated genes to gain or loss new regulatory elements simultaneously. Overall, several of duplicated ohnologs have similar transcription profiles in response to pro-growth signals suggesting that evolution tends to conserve ohnolog regulation during muscle development and that in the majority of ohnologs related to muscle growth their functions might be very similar.

Influence of Low Dietary Inclusion of the Microalga Nannochloropsis gaditana (Lubián 1982) on Performance, Fish Morphology, and Muscle Growth in Juvenile Gilthead Seabream (Sparus aurata).

A 90-d feeding trial was conducted in which five groups of gilthead seabream (11.96 g initial body weight) were fed with a microalgae-free diet (control group, C) or four diets containing the microalgae Nannochloropsis gaditana at two inclusion levels (2.5% or 5%), either raw (R2.5 and R5 batches) or cellulose-hydrolyzed (H2.5 and H5 batches), to study their effect on the body and muscle growth. At 40 days, the highest values of body length and weight were reached in R5 group, but at 64 and 90 days, these were reached in R2.5. However, feed conversion rate, specific growth, daily intake, and survival (100%) were similar in all the groups. The acquisition of a discoid body shape was accelerated depending on the inclusion level of N. gaditana in the diets. Moreover, H5 diet affected the fish geometric morphology compared to R5 diet. The white muscle transverse area was similar in all groups at 40 days, with the exception of H2.5 group, which showed the lowest area. At day 90, C and R2.5 displayed the highest muscle growth, attributable to increased hyperplasia in C, and higher hypertrophy in R2.5. However, the highest proportion of small and medium fibers was observed in R5 and H5.

Rainbow trout slow myoblast cell culture as a model to study slow skeletal muscle, and the characterization of mir-133 and mir-499 families as a case study.

Muscle fibres are classified as fast, intermediate and slow. In vitro myoblast cell culture model from fast muscle is a very useful tool to study muscle growth and development; however, similar models for slow muscle do not exist. Owing to the compartmentalization of fish muscle fibres, we have developed a slow myoblast cell culture for rainbow trout (Oncorhynchus mykiss). Slow and fast muscle-derived myoblasts have similar morphology, but with differential expression of slow muscle markers such as slow myhc, sox6 and pgc-1α We also characterized the mir-133 and mir-499 microRNA families in trout slow and fast myoblasts as a case study during myogenesis and in response to electrostimulation. Three mir-133 (a-1a, a-1b and a-2) and four mir-499 (aa, ab, ba and bb) paralogues were identified for rainbow trout and named base on their phylogenetic relationship to zebrafish and Atlantic salmon orthologues. Omy-mir-499ab and omy-mir-499bb had 0.6 and 0.5-fold higher expression in slow myoblasts compared with fast myoblasts, whereas mir-133 duplicates had similar levels in both phenotypes and little variation during development. Slow myoblasts also showed increased expression for omy-mir-499b paralogues in response to chronic electrostimulation (7-fold increase for omy-mir-499ba and 2.5-fold increase for omy-mir-499bb). The higher expression of mir-499 paralogues in slow myoblasts suggests a role in phenotype determination, while the lack of significant differences of mir-133 copies during culture development might indicate a different role in fish compared with mammals. We have also found signs of sub-functionalization of mir-499 paralogues after electrostimulation, with omy-mir-499b copies more responsive to electrical signals.

Duplication of a Single myhz1.1 Gene Facilitated the Ability of Goldfish (Carassius auratus) to Alter Fast Muscle Contractile Properties With Seasonal Temperature Change.

Seasonal temperature changes markedly effect the swimming performance of some cyprinid fish acutely tested at different temperatures, involving a restructuring of skeletal muscle phenotype including changes in contractile properties and myosin heavy chain expression. We analyzed the transcriptome of fast myotomal muscle from goldfish (Carassius auratus L.) acclimated to either 8 or 25°C for 4 weeks (12 h light: 12 h dark) and identified 10 myosin heavy chains (myh) and 13 myosin light chain (myl) transcripts. Goldfish orthologs were classified based on zebrafish nomenclature as myhz1.1α, myhz1.1ß, myhz1.1γ, myha, myhb, embryo_myh1, myh9b, smyh2, symh3, and myh11 (myosin heavy chains) and myl1a, myl1b, myl2, myl9a, myl9b, myl3, myl13, myl6, myl12.1a, myl12.1b, myl12.2a, myl12.2b, and myl10 (myosin light chains). The most abundantly expressed transcripts myhz1.1α, myhz1.1ß, myhz1.1γ, myha, myl1a, myl1b, myl2, and myl3) were further investigated in fast skeletal muscle of goldfish acclimated to either 4, 8, 15, or 30°C for 12 weeks (12 h light:12 h dark). Total copy number for the myosin heavy chains showed a distinct optimum at 15°C (P < 0.01). Together myhz1.1α and myhz1.1ß comprised 90 to 97% of myhc transcripts below 15°C, but only 62% at 30°C. Whereas myhz1.1α and myhz1.1ß were equally abundant at 4 and 8°C, myhz1.1ß transcripts were 17 and 12 times higher than myhz1.1α at 15 and 30°C, respectively, (P < 0.01). Myhz1.1γ expression was at least nine-fold higher at 30°C than at cooler temperatures (P < 0.01). In contrast, the expression of myha and myosin light chains showed no consistent pattern with acclimation temperature. A phylogenetic analysis indicated that the previously reported ability of goldfish and common carp to alter contractile properties and myofibrillar ATPase activity with temperature acclimation was related to the duplication of a single myhz1.1 fast muscle myosin heavy chain found in basal cyprinids such as the zebrafish (Danio rerio).

Insulin-Like Growth Factor-Binding Proteins of Teleost Fishes.

The insulin-like growth factor (Igf) binding protein (Igfbp) family has a broad range of physiological functions and a fascinating evolutionary history. This review focuses on the Igfbps of teleost fishes, where genome duplication events have diversified gene repertoire, function, and physiological regulation-with six core Igfbps expanded into a family of over twenty genes in some lineages. In addition to briefly summarizing the current state of knowledge on teleost Igfbp evolution, function, and expression-level regulation, we highlight gaps in our understanding and promising areas for future work.

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.

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.

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.

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.