Integrative Metabolomics, Enzymatic Activity, and Gene Expression Analysis Provide Insights into the Metabolic Profile Differences between the Slow-Twitch Muscle and Fast-Twitch Muscle of Pseudocaranx dentex.

The skeletal muscles of teleost fish encompass heterogeneous muscle types, termed slow-twitch muscle (SM) and fast-twitch muscle (FM), characterized by distinct morphological, anatomical, histological, biochemical, and physiological attributes, driving different swimming behaviors. Despite the central role of metabolism in regulating skeletal muscle types and functions, comprehensive metabolomics investigations focusing on the metabolic differences between these muscle types are lacking. To reveal the differences in metabolic characteristics between the SM and FM of teleost, we conducted an untargeted metabolomics analysis using Pseudocaranx dentex as a representative model and identified 411 differential metabolites (DFMs), of which 345 exhibited higher contents in SM and 66 in FM. KEGG enrichment analysis showed that these DFMs were enriched in the metabolic processes of lipids, amino acids, carbohydrates, purines, and vitamins, suggesting that there were significant differences between the SM and FM in multiple metabolic pathways, especially in the metabolism of energy substances. Furthermore, an integrative analysis of metabolite contents, enzymatic activity assays, and gene expression levels involved in ATP-PCr phosphate, anaerobic glycolysis, and aerobic oxidative energy systems was performed to explore the potential regulatory mechanisms of energy metabolism differences. The results unveiled a set of differential metabolites, enzymes, and genes between the SM and FM, providing compelling molecular evidence of the FM achieving a higher anaerobic energy supply capacity through the ATP-PCr phosphate and glycolysis energy systems, while the SM obtains greater energy supply capacity via aerobic oxidation. These findings significantly advance our understanding of the metabolic profiles and related regulatory mechanisms of skeletal muscles, thereby expanding the knowledge of metabolic physiology and ecological adaptation in teleost fish.

Cloning, expression profiling and promoter functional analysis of Bone morphogenetic protein 6 and 7 in tongue sole (Cynoglossus semilaevis).

Bone morphogenetic proteins (BMPs) play crucial roles in vertebrate developmental process and are associated with the mechanisms which drive early skeletal development. As a first approach to elucidating the role of BMPs in regulating fish bone formation and growth, we describe the cloning, expression profiling and promoter functional analysis of bmp6 and bmp7 in tongue sole (Cynoglossus semilaevis). The full length of bmp6 and bmp7 cDNA sequences is 1939 and 1836 bp, which encodes a protein of 428 and 427 amino acids, respectively. Tissue expression distribution of bmp6 and bmp7 was examined in 14 tissues of mature individuals by quantitative real-time PCR (qRT-PCR). The results revealed that bmp6 was predominantly expressed in the gonad, and bmp7 exhibited the highest expression level in the dorsal fin. Further comparison of bmp6 expression levels between female and male gonads showed that the expression in the ovary was significantly higher than in the testis. Moreover, bmp6 and bmp7 expression levels were detected at 15 sampling time points of early developmental stages (egg, larva, juvenile and fingerling stages). The highest expression level of bmp6 was observed in the egg stage (multi-cell and gastrula stage); while bmp7 exhibited the highest expression in the larva stage (1-4 days old). The high expression levels of BMP6 in the ovary as well as at early embryonic stages indicated that the maternally stored transcripts of bmp6 might play a role in early embryonic development. Whole-mount in situ hybridization showed that bmp6 and bmp7 exhibited similar spatial expression patterns. Both bmp6 and bmp7 signals were first detected in the head and anterior regions in newly hatched larvae, and then, the mRNAs appeared in the crown-like larval fin, jaw, operculum and fins (pectoral, dorsal, pelvic and anal) along with early development. Subsequently, we characterized the 5'-flanking regions of bmp6 and bmp7 by testing the promoter activity by luciferase reporter assays. Positive regulatory regions were, respectively, detected at the location of -272 to +28 and -740 to -396 in bmp6 and bmp7 gene. The predicted transcription factor binding sites (CREB, AP1 and methyl-CpG-binding protein) in the regions might participate in the transcriptional regulation of these two genes.

Expression profiles and transcript properties of fast-twitch and slow-twitch muscles in a deep-sea highly migratory fish, Pseudocaranx dentex.

Fast-twitch and slow-twitch muscles are the two principal skeletal muscle types in teleost with obvious differences in metabolic and contractile phenotypes. The molecular mechanisms that control and maintain the different muscle types remain unclear yet. Pseudocaranx dentex is a highly mobile active pelagic fish with distinctly differentiated fast-twitch and slow-twitch muscles. Meanwhile, P. dentex has become a potential target species for deep-sea aquaculture because of its considerable economic value. To elucidate the molecular characteristics in the two muscle types of P. dentex, we generated 122 million and 130 million clean reads from fast-twitch and slow-witch muscles using RNA-Seq, respectively. Comparative transcriptome analysis revealed that 2,862 genes were differentially expressed. According to GO and KEGG analysis, the differentially expressed genes (DEGs) were mainly enriched in energy metabolism and skeletal muscle structure related pathways. Difference in the expression levels of specific genes for glycolytic and lipolysis provided molecular evidence for the differences in energy metabolic pathway between fast-twitch and slow-twitch muscles of P. dentex. Numerous genes encoding key enzymes of mitochondrial oxidative phosphorylation pathway were significantly upregulated at the mRNA expression level suggested slow-twitch muscle had a higher oxidative phosphorylation to ensure more energy supply. Meanwhile, expression patterns of the main skeletal muscle developmental genes were characterized, and the expression signatures of Sox8, Myod1, Calpain-3, Myogenin, and five insulin-like growth factors indicated that more myogenic cells of fast-twitch muscle in the differentiating state. The analysis of important skeletal muscle structural genes showed that muscle type-specific expression of myosin, troponin and tropomyosin may lead to the phenotypic structure differentiation. RT-qPCR analysis of twelve DEGs showed a good correlation with the transcriptome data and confirmed the reliability of the results presented in the study. The large-scale transcriptomic data generated in this study provided an overall insight into the thorough gene expression profiles of skeletal muscle in a highly mobile active pelagic fish, which could be valuable for further studies on molecular mechanisms responsible for the diversity and function of skeletal muscle.

Complete Mitochondrial Genome of Pseudocaranx dentex (Carangidae, Perciformes) Provides Insight into Phylogenetic and Evolutionary Relationship among Carangidae Family.

Pseudocaranx dentex (white trevally) which belongs to the Carangidae family, is an important commercial fishery and aquaculture resource in Asia. However, its evolution and population genetics have received little attention which was limited by the mitogenome information absence. Here, we sequenced and annotated the complete mitochondrial genome of P. dentex which was 16,569 bp in length, containing twenty-two tRNAs (transfer RNAs), thirteen PCGs (protein-coding genes), two rRNAs (ribosomal RNAs), and one non-coding region with conservative gene arrangement. The Ka/Ks ratio analysis among Carangidae fishes indicated the PCGs were suffering purify selection and the values were related to the taxonomic status and further influenced by their living habits. Phylogenetic analysis based on the PCGs sequences of mitogenomes among 36 species presented three major clades in Carangidae. According to the phylogenetic tree, we further analyzed the taxonomic confusion of Carangoides equula which was on the same branch with P. dentex but a different branch with Carangoides spp. We inferred Kaiwarinus equula should be the accepted name and belong to the independent Kaiwarinus genus which was the sister genus of Pseudocaranx. This work provides mitochondrial genetic information and verifies the taxonomic status of P. dentex, and further helps to recognize the phylogenetic relationship and evolutionary history of Carangidae.