- Research article
- Open Access
Characterization of the transcriptome of fast and slow muscle myotomal fibres in the pacu (Piaractus mesopotamicus)
© Mareco et al.; licensee BioMed Central. 2015
- Received: 1 August 2014
- Accepted: 28 February 2015
- Published: 14 March 2015
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.
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.
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.
- Ubiquitin-specific proteases (USP)
- Aquaculture genomics
Fish myotomes are composed of anatomically segregated muscle fibre types each with distinct contractile and metabolic phenotypes . Based on their contractile speed skeletal muscle fibres are classified as slow, intermediate or fast [2,3]. Fast twitch (white) muscle fibres comprise the bulk of the myotome and are recruited for energetic movements associated with prey capture and escape behaviour . Fast fibres have elevated densities of myofibrils, reduced myoglobin content and higher a capillary density than slow fibres and utilise phosphagen breakdown and anaerobic glycolysis to power contraction . Sustained swimming activity is supported by superficial layers of slow (red) and intermediate (pink) twitch fibres which are recruited at slow and high cruising speeds respectively . Slow fibres have extensive lipid and glycogen stores, abundant mitochondria and high capillary densities reflecting their reliance on aerobic metabolism . Intermediate (pink) muscle fibre types are found between the slow and fast fibre layers, but express distinct isoforms of myosin heavy chains  and have intermediate contraction speeds and metabolic characteristics [3,7].
Muscle growth reflects the balance between protein synthesis and degradation. These two processes are influenced by numerous biotic and abiotic factors including food availability, growth factors, age, sex, diet composition, swimming activity, oxygen saturation, light and temperature [8,9]. The Insulin-like growth factor (Igf) network, composed of Igfs, binding proteins (Igfbp) and receptors (Igf1r and Igf2r), plays a pivotal role in integrating internal and external inputs to regulate muscle mass . Igf1 regulates several signalling pathways including the Pi3k/Akt/mTor network that controls protein synthesis [8,9]. Typically, fibre production continues until 45% of the maximum body length of the fish, and subsequent growth is entirely by fibre elongation and hypertrophy [10-12]. Myogenesis involves the activation, proliferation and fusion of a resident myoblast population involving hundreds of structural and regulatory genes [10,11].
Muscle protein degradation occurs through three major pathways [13,14] namely: membrane-bound lysosomal enzymes, calpain proteases [14,15], and the Ubiquitin Proteasome (Ub) Pathway (UPP) . UPP represents the most important system for degradation of unnecessary or damaged proteins. Targeted proteins are linked to ubiquitin, which acts as a recognition tag for the proteasome. Ubiquitin tagging of proteins requires the coordinated activity of three classes of enzymes known as E1, E2 and E3. It is the E3 enzymes, also known as E3-ubiquitin ligases, which conjugate ubiquitin to the target protein . Ubiquitin mediated degradation can be reversed through action of deubiquitinating enzymes (DUB), a large group of proteases that cleave ubiquitin-protein conjugates removing the UPP signal, and play an essential role in the regulation of protein degradation . DUBs are subdivided into four families: ubiquitin C-terminal hydrolases (UCHs), ovarian tumour proteases (OTUs), JAB1/MPN/MOV34 metalloenzymes and ubiquitin-specific proteases (USPs) . USP is the largest family of DUB and regulates a wide variety of cellular processes. Although the essential role of USPs in protein degradation is well established, less is known about the function and regulation of specific family members: for example Usp7 has been associated with p53 and Akt turnover, Usp8 with receptor tyrosine endocytosis, Usp33 with the Von Hippel–Lindau disease (VHL) pathway and Usp19 is thought to have a role in muscle development . There have been several studies of the expression of protein degradation related genes in fish, mostly in response to varying nutrition and focused on E3-ubiquitin ligases (particularly Fbox32 and Murf1) [20,21]. In contrast, nothing is known about the transcriptional regulation of USP family members.
The pacu (Piaractus mesopotamicus) is a member of the characiform family (SuperOder Ostariophysi) native to the Prata Basin (South America) and is a target for finfish aquaculture in Brazil. One of the main limitations for the development of a selective breeding program for this species is a lack of genetic information as well as limited knowledge about its physiology. Next Generation Sequencing (NGS) technologies have dramatically increased the amount of sequence data for teleosts and helped to overcome the lack of annotated genomes (so far only 12 fish annotated genomes are publicly available) .
The primary objectives of the present study were 1) to increase the genetic resources available for pacu 2) to exploit the anatomical separation of fibre types to characterise the expression signatures of fast and slow muscles and 3) to systematically investigate the expression of USPs in fast and slow muscle in response to fasting and refeeding. Teleost fish underwent a teleost-specific genome duplication (TSGD) event around 450 million years ago (Mya) which was followed by diploidisation and gene loss [23,24]. It is thought that around 15 to 20% of TSGD paralogues have been retained in the diploid genome of extant species [23-26]. Several studies have demonstrated that paralogues from the TSGD or the salmonid-specific whole-genome duplication (WGD), which occurred 88Mya , can display different patterns of expression during myogenesis and muscle growth [27-29]. A secondary objective was therefore to search for evidence of differential expression between teleost specific paralogues within and between muscle fibre types.
De novo assembly
Pacu de novo transcriptome metrics
Slow skeletal muscle
Fast skeletal muscle
Contig mean lenght (bp)
Differentially expressed genes
Digital gene expression analysis
Gene ontology (GO) enrichment analysis
Genes in category
Generation of precursor metabolites and energy
Anatomical structure morphogenesis
Structural molecule activity
Multicellular organismal development
Calcium ion binding
Ion channel activity
Lipid metabolic process
Electron carrier activity
Carbohydrate metabolic process
Generation of precursor metabolites and energy
Carbohydrate metabolic process
Organonitrogen compound metabolic process
Carboxylic acid metabolic process
Ion channel activity
Calcium ion binding
Transcriptional regulation of USP and E3-ubiquitin ligases
The E3-ubiquitin ligases are an essential part of the proteasome system, directly involved in protein degradation [45,46]. All E3-ubiquitin ligases increased their relative abundance during fasting, for example, fbox-32 a muscle specific E3-ubiquitin ligase increased 100-fold (Figure 5B) and fbox25 increased 10-fold, in line with results from other similar studies [21,47]. The majority of the USPs increased their expression around 2-fold with fasting and recovered pre-fasting levels 24 h after re-feeding (Figure 5A). In contrast, Usp12a, showed a transient increase 6 h after re-feeding, and usp46 and usp5b did not change in expression (Figure 5D and E). The overlapping expression profiles of USPs and E3-ubiquitin ligases suggests that USPs play an important role during muscle atrophy. USPs can cleave ubiquitin from proteins, effectively removing the proteasome signal . It is possible that the increase in their abundance in fasted fish is related to fine tuning of the regulation of protein degradation. For example, many USPs targets are essential to maintain cell homeostasis including mdm4, p53, h2a, h2b, fbw7, fancd2 or brca2 [17,48-50], and it is possible that these proteins may be relatively spared during fasting.
We have produced an in depth transcriptome for fast and slow myotomal muscle for the pacu (Piaractus mesopotamicus), an important South American aquaculture species. This resource allowed us to characterise the expression signatures of the main myotomal muscle fibre types and identify candidate microsatellite sequences that could be used in breeding programs. The availability of the transcriptome allowed us to identify and analyse the expression of teleost-specific paralogues retained in the Ostariophysi lineage. The transcriptome also enabled a comprehensive study of E3 ubiquitin ligase and USP gene expression in the context of the transition between the fasting (catabolic state) and satiation feeding (anabolic state). We identified differences in expression within gene family members thereby identifying candidates for further investigation.
For the generation of de novo transcriptome fast and slow skeletal muscles were dissected from 5 adult pacu (Piaractus mesopotamicus) (1.50 ± 0.61 kg; mean ± SD body mass). Fish were maintained in 1000 litres fibreglass tanks at the Aquaculture Centre of the University of West Paulista (Unoeste) Presidente Prudente, São Paulo, Brazil, under natural photoperiod (12 L: 12D) and temperature (28°C ± 1°C, range). Fast skeletal muscle was dissected from the dorsal epaxial region at 0.5 fork length (FL) (FL, tip of snout to fork in the tail) whereas slow skeletal muscle was dissected from the lateral line and any remains of fast skeletal muscle carefully removed under a dissection microscope to obtain pure slow muscle. Tissues were preserve in RNAlater (Ambion/Applied Biosystems, Oslo, Norway) and frozen at −20°C until further analysis.
For the fasting-re-feeding experiment, 15 g pacu (n = 80) were maintained in duplicate fibreglass 500 litre tanks as described above and fed with a commercial diet until the start the experiment. Fish were fasted for 4 days followed by a period of satiation feeding for 24 h. Fast skeletal muscle was sampled before fasting (−4d), daily during food deprivation (−3d, −2d, −1d, 0d; n = 8) and 6, 12 and 24 h (n = 8) after re-feeding. Dissected fast skeletal muscle was preserved in RNAlater at −20°C until further analysis. All fish were sacrificed according to the Ethical Principles In Animal Research adopted by Brazilian College of Animal Experimentation (COBEA) and was approved by the Ethics Committee on Use of Animals/ Bioscience Institute/Unesp (CEUA = 506).
Samples sequencing and de novo assembly
Total RNA from adult pacu fast and slow skeletal muscle was used to prepare 10 individual Illumina libraries. Libraries preparation and sequencing was performed at the Centre for Applied Genomics of the Hospital for Sick Children (SickKids), Toronto, Canada. The resulting libraries were paired end sequenced using in an Illumina HiSeq2000.
Raw paired end reads generated were processed by the Department of Informatics of the Centre for Applied Genomics of SickKids hospital. After removing low quality reads, 86% of the paired end reads were de novo assembled using Trinity software . RSEM application was used to identify transcript abundance by estimating the number of reads mapped per contig. The DEseq algorithm from the Bioconductor/R packages was used to identify differentially expressed transcripts .
Contigs were annotated using Blast2GO software . Sequences were blasted against the NCBI non-redundant (nr) database using BLASTx with an e-value cut-off of 10−3 followed by functional annotation using software default parameters . Contigs were mapped against the known vertebrate metabolic and molecular pathways using the online KEGG Automatic Annotation Server (KAAS) . KAAS annotation was performed using the single-directional Best Hit (SBH) method against Homo sapiens, Pan troglodites, Mus musculus, Rattus norvegicus, Sus scrofa, Gallus gallus, Meleagris gallopavo, Danio rerio and Xenopus laevis.
Identification of complete coding sequence
Annotated contigs were blasted against the Zebrafish complete proteome  using tBLASTn algorithm in BioEdit software . BLAST alignments were explored to evaluate the percentage of conding sequence cover by the contig compared with its zebrafish orthologue. Sequences with more than 90% of coverage were considered as complete coding sequence (CDS). The CDS amino acids sequence was predicted using the Virtual Ribosome server .
Sequences successfully annotated covering >90% of the CDS were investigated for SSR using msatcommander-1.0.2-alpha .
RNA Extraction and cDNA synthesis
Total RNA was extracted using 1 ml TRIsure (Bioline, London, UK) following the manufacturer’s recommendations. Integrity was confirmed by ethidium bromide gel electrophoresis of 1 μg of total RNA. Concentration, 260/280 and 260/230 ratios were estimated using a NanoDrop 1000 spectrophotometer (Thermo Fischer Scientific, Waltman, MA). All RNA samples used had 260/280 nm and 260/230 ratios above 1.9 and 2.2 respectively. 1 μg of total RNA was reverse transcribed into cDNA for 30 min at 42°C using a Quantitect (QIAGEN, Manchester, UK) reverse transcription kit following manufacture’s recommendations including a genomic DNA wipe-out step. To ensure that no genomic DNA was present in the samples a RT- control without the reverse transcriptase enzyme was performed simultaneously.
Quantitative Real-time PCR
The following procedures were compliant with the minimal information requirements for publication of quantitative PCR guidelines . Primers were designed to have a Tm of 60°C using Net primer online server (Premier BioSoft). Where possible primers were designed to cross exon-exon junctions. Exon-exon junctions were predicted by aligning the pacu contig against their zebrafish orthologue complete gene sequence retrieved from Ensembl  using Spidey online server . Primers pairs, amplicon size and efficiency are listed in Additional file 12.
Quantitative PCR (qPCR) was performed using a MX3005P qPCR machine (Agilent, La Jolla, CA, USA). Each qPCR reaction contained 7.5 μl of SensiFast (Bioline) Master Mix, 6 μl cDNA (80-fold dilution and 40-fold dilution for igf genes) and 0.75 μl of each primer at 500 nM to a final volume of 15 μl. Duplicate reactions were performed in 96-well plates (Agilent) with the following protocol: initial activation 95°C for 2 min followed by 40 cycles of 5 s at 95°C, 20s at 65°C. The qPCR was followed by a dissociation-melting curve from 60 to 95°C to confirm that a single product was amplified. Control reactions included no-template and RT- were simultaneously amplified.
Ribosomal protein 13 and 19 (rpl13, rpl19), Peptidylprolyl isomerase Aa (ppiaa), elongation factor 1 alpha, Glyceraldehyde-3-phosphate dehydrogenase (gapdh) and hypoxanthine phosphoribosyltransferase 1 (hprt1) were tested as reference genes. BestKeeper  analysis showed that hprt1 was the most stable reference gene and was used for data normalisation. Relative expression was calculated using the Pfalff method .
Teleost specific paralogues identification
Contigs were blasted (BLASTx) against the Zebrafish proteome using BioEdit software with an e-value threshold of e−40 .
To confirm that contigs found were truly paralogues amino acids sequences from potential pacu paralogues were blasted (BLASTp) against the zebrafish (Danio rerio), stickleback (Gasterosteus aculeatus), takifugu (Takifugu rubripes), medaka (Oryzias latipes), green pufferfish (Tetraodon nigroviridis), chicken (Gallus gallus), frog (Xenopus laevis) and human (Homo sapiens) proteomes using Ensembl BLAST server . Best hits amino acids sequences from each proteome were retrieved. Sequences were aligned using the MAFFT online server . Phylogenetic trees were constructed using a Maximum Likelihood analysis using PhyML online server combined with the G-Blocks option to cure unreliable aligned sections . For each case the best evolutionary model was estimated using MEGA5 .
Global DGE statistic analysis was performed using DEseq package from R-Bioconductor [52,65]. For testing specific hypothesis involving differential mapping of specific pathways gene expression significance was tested using t-test, or Mann–Whitney U test when parametric were not fulfilled, followed by a Benjamin-Hochberg correction (False Discovery Rate, FDR). Differences between time-points in qPCR expression during fasting re-feeding were tested using Kruskal-Wallis test. Differences were considered significant when FDR, for differential mapping, or p-value, for qPCR expression, were <0.05. Gene expression data was clustered using an unsupervised hierarchical clustering algorithm using PermutMatrix .
MDPS and EAM were supported by FAPESP, Proc. n° 12/02489-4 and 2011/09346-1. This work also received funding from CAPES, Proc. n° 2524/12. IAJ and DGDLS were supported by the Marine Alliance for Science and Technology for Scotland pooling initiative and Scottish Funding Council grant number HR09011.
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