Morphological and Molecular Responses of Lateolabrax maculatus Skeletal Muscle Cells to Different Temperatures.

Temperature strongly modulates muscle development and growth in ectothermic teleosts; however, the underlying mechanisms remain largely unknown. In this study, primary cultures of skeletal muscle cells of Lateolabrax maculatus were conducted and reared at different temperatures (21, 25, and 28 °C) in both the proliferation and differentiation stages. CCK-8, EdU, wound scratch and nuclear fusion index assays revealed that the proliferation, myogenic differentiation, and migration processes of skeletal muscle cells were significantly accelerated as the temperature raises. Based on the GO, GSEA, and WGCNA, higher temperature (28 °C) induced genes involved in HSF1 activation, DNA replication, and ECM organization processes at the proliferation stage, as well as HSF1 activation, calcium activity regulation, myogenic differentiation, and myoblast fusion, and sarcomere assembly processes at the differentiation stage. In contrast, lower temperature (21 °C) increased the expression levels of genes associated with DNA damage, DNA repair and apoptosis processes at the proliferation stage, and cytokine signaling and neutrophil degranulation processes at the differentiation stage. Additionally, we screened several hub genes regulating myogenesis processes. Our results could facilitate the understanding of the regulatory mechanism of temperature on fish skeletal muscle growth and further contribute to utilizing rational management strategies and promoting organism growth and development.

Methylation status and expression patterns of myomaker gene play important roles in postnatal development in the Japanese flounder (Paralichthys olivaceus).

Myomaker is a membrane protein that plays a crucial role in the fusion of myoblasts during muscle growth. DNA methylation, a significant factor, regulates gene expression. The aim of this study was to examine the methylation and mRNA expression patterns of the myomaker gene during 8 different postnatal developmental stages in the Japanese flounder (L: 7 days post hatch (dph); M1: 21 dph; M2: 28 dph; M3: 35 dph; J1: 90 dph; J2: 180 dph; A1: 24 months; A2: 36 months). Muscle tissue samples were taken from Japanese flounder at different postnatal development stages to measure the extent of DNA methylation and gene expression. Methylation level in the promoter and exon 1 of myomaker was measured using bisulfite sequencing, and the relative expression of myomaker during each developmental stage was measured by quantitative PCR. The relative expression levels of myomaker were up-regulated from stages L to M2, M3 to J2, and methylation of myomaker was negatively correlated with mRNA expression. Furthermore, the CpG site located at -26 bp in the promoter was the lowest methylated region in all developmental stages. These results offer a basis for understanding the mechanism by which myomaker regulates muscle formation during postnatal development.

DNA methylation in promoter region of immune related genes STAT3 and VEGFA and biochemical parameters change in muscle of Japanese flounder under acute hypoxia.

Acute hypoxic stress can lead to immune response in fish, but the molecular mechanism of muscle immunity in fish under acute hypoxia are still unclear. In this study, we carried out the effect of signal transducer and activator of transcription3(STAT3) and vascular endothelial growth factor A(VEGFA) on muscle immune responses of Japanese flounder (Paralichthys olivaceus) during acute hypoxic stimulation (1.65 ± 0.28mg/L O2; 3h, 6h, 12h, 24h) and reoxygenation (7.30 ± 0.40mg/L O2; R12h, R24h, R48h). In situ hybridization (ISH) showed that STAT3 and VEGFA RNA were co-located in the skeletal muscle of Japanese flounder. Japanese flounder was seriously affected by hypoxia for 3h and 6h. The expression of STAT3 and VEGFA increased significantly. The methylation levels of STAT3 5'UTR region and VEGFA promoter region were significantly lower than those in normoxia group, which was negatively correlated with the expression levels of STAT3 and VEGFA. The enzyme activities (LDH, ALT, AST, ALP) changed significantly. In addition, enzyme-linked immunosorbent assay (ELISA) detected a positive correlation between serum VEGFA concentration and muscle VEGFA mRNA. The current study have shown that Japanese flounder responded to acute hypoxic stress at multiple metabolic levels by changing DNA methylation status and activating transcription factors such as HIF-1α, Nrf2 and STAT3. It is significant for the scientific development of aquaculture through analyzing the effects of hypoxia on biological immunity.

Acute hypoxia effects on Keap1/Nrf2 (Mafs)-GST pathway related oxidative metabolism in muscle of Japanese flounder (Paralichthys olivaceus).

Acute hypoxia can aggravate the oxidation metabolism of fish muscle tissue. However, the molecular mechanism of oxidative metabolism in fish muscle under acute hypoxia is not very clear. We carried out effects of a typical oxidative metabolism pathway Keap1/Nrf2 (MafG)-GST on muscle oxidative metabolism of Japanese flounder (Paralichthys olivaceus) during acute hypoxia stimulation (1.65 ± 0.05 mg/L; 1 h, 3 h, 6 h, 12 h, 24 h) and reoxygenation (7.30 ± 0.08 mg/L; R12 h, R24 h, R48 h). The mRNAs of Nrf2 and GST in skeletal muscle were found co-existent, and their expressions were significant increase in 3 h and 6 h. The methylation level of CpG island1 in Nrf2 promoter, whose minimum value appeared at 3 h hypoxia treatment group, was affected by acute hypoxia, and it was negatively correlated with Nrf2 expression. The result suggests that environmental factors may regulate gene expression by epigenetic modification. Dual-luciferase reporter assay showed that GST gene was activated by transcription factor Nrf2, whose transcriptional activation binding region in GST promoter was antioxidant response element located near -980 and -852 sites, and Keap1 and MafG were Nrf2 antagonistic and synergistic factor, respectively. Furthermore, the GST activity changed with hypoxia and reoxygenation treatment in muscle, where other oxidative stress factor (MDA), antioxidant factors (T-AOC, GSH) and antioxidant enzyme activities (GST, SOD, CAT) were also changed. The results of MDA and T-AOC being further different between its hypoxia and normoxia groups (P < 0.05) at 6 h demonstrated that hypoxia stimulation lasting for 6 h would deeply affect Japanese flounder. The study illustrated that Japanese flounder responded to acute hypoxia in multiple metabolic levels by changing methylation status and transcription factor activation. It is significant to understand oxidative metabolic mechanism, analyze organism stress response and promote the scientific development of aquaculture.

Transcriptional Profiles of Genes Related to Stress and Immune Response in Rainbow Trout (Oncorhynchus mykiss) Symptomatically or Asymptomatically Infected With Vibrio anguillarum.

Rainbow trout (Oncorhynchus mykiss) is one of the most common aquaculture fish species worldwide. Vibriosis disease outbreaks cause significant setbacks to aquaculture. The stress and immune responses are bidirectionally modulated in response to the health challenges. Therefore, an investigation into the regulatory mechanisms of the stress and immune responses in trout is invaluable for identifying potential vibriosis treatments. We investigated the transcriptional profiles of genes associated with stress and trout immune functions after Vibrio anguillarum infection. We compared the control trout (CT, 0.9% saline injection), asymptomatic trout (AT, surviving trout with minor or no symptoms after bacteria injection), and symptomatic trout (ST, moribund trout with severe symptoms after bacteria injection). Our results showed activated immunomodulatory genes in the cytokine network and downregulated glucocorticoid and mineralocorticoid receptors in both AT and ST, indicating activation of the proinflammatory cytokine cascade as a common response in AT and ST. Moreover, the AT specifically activated the complement- and TNF-associated immune defenses in response to V. anguillarum infection. However, the complement and coagulation cascades, as well as steroid hormone homeostasis in ST, were disturbed by V. anguillarum. Our studies provide new insights toward understanding regulatory mechanisms in stress and immune functions in response to diseases.

Identification and Characterization of lncRNAs Related to the Muscle Growth and Development of Japanese Flounder (Paralichthys olivaceus).

Long noncoding RNAs (lncRNAs) play an important role in many life activities, but the expression pattern and function of lncRNAs in Japanese flounder skeletal muscle are unclear. In this study, 751 lncRNAs were selected from skeletal muscle in different development stages of the Japanese flounder [stage A: larval 7 days post hatching (dph); stage B: juvenile about 90 dph; stage C (female) and stage D (male): adult about 24 months] using coding potential analysis methods. In total, 232, 211, 194, 28, 29, and 14 differentially expressed lncRNAs and 9549, 8673, 9181, 1821, 1080, and 557 differentially expressed mRNAs were identified in comparisons of A versus B, A versus C, A versus D, B versus C, B versus D, and C versus D, respectively. We identified the cis- and trans-regulatory target genes of differentially expressed lncRNAs, and lncRNA-gene interaction networks were constructed using the Cytoscape program. In total, there were 200, 200, 200, 93, 47, and 11 cis-regulation relationships, and 29, 19, 24, 38, 8, and 47 trans-regulation relationships in the comparisons between A versus B, A versus C, A versus D, B versus C, B versus D, and C versus D, respectively. These results indicate that lncRNA may participate in the development of Japanese flounder skeletal muscle through cis- or trans-acting mechanisms, thus providing a scientific basis for further study of the biological function of lncRNA in Japanese flounder skeletal muscle. Based on these relationships, functional annotation of the related lncRNAs was performed by gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Differentially expressed genes associated with muscle development were enriched in multiple pairs of comparisons (e.g., differentially expressed genes LOC109640370, LOC109634180, LOC109643555, rusc1, and LOC109626999 were enriched in the actin-binding term, and differentially expressed genes LOC109640370, was, LOC109644970, LOC109643555, and itga9 were enriched in the regulation of the actin cytoskeleton pathway in the KEGG pathway analysis in the comparison of stages C and D). We predicted lncRNA-mRNA, miRNA-mRNA, and lncRNA-miRNA regulatory relationships and constructed interactive networks using Cytoscape software. Co-expression networks show that most lncRNAs interact with one or two predicted miRNAs involved in muscle growth and development. These results provide a basis for further study of the function of lncRNAs on skeletal muscle in different developmental stages of Japanese flounder.

First High-Density Linkage Map and QTL Fine Mapping for Growth-Related Traits of Spotted Sea bass (Lateolabrax maculatus).

Possessing powerful adaptive capacity and a pleasant taste, spotted sea bass (Lateolabrax maculatus) has a broad natural distribution and is one of the most popular mariculture fish in China. However, the genetic improvement program for this fish is still in its infancy. Growth is the most economically important trait and is controlled by quantitative trait loci (QTL); thus, the identification of QTLs and genetic markers for growth-related traits is an essential step for the establishment of marker-assisted selection (MAS) breeding programs. In this study, we report the first high-density linkage map of spotted sea bass constructed by sequencing 333 F1 generation individuals in a full-sib family using 2b-RAD technology. A total of 6883 SNP markers were anchored onto 24 linkage groups, spanning 2189.96 cM with an average marker interval of 0.33 cM. Twenty-four growth-related QTLs, including 13 QTLs for body weight and 11 QTLs for body length, were successfully detected, with phenotypic variance explained (PVE) ranging from 5.1 to 8.6%. Thirty potential candidate growth-related genes surrounding the associated SNPs were involved in cell adhesion, cell proliferation, cytoskeleton reorganization, calcium channels, and neuromodulation. Notably, the fgfr4 gene was detected in the most significant QTL; this gene plays a pivotal role in myogenesis and bone growth. The results of this study may facilitate marker-assisted selection for breeding populations and establish the foundation for further genomic and genetic studies investigating spotted sea bass.

Alternative splicing (AS) mechanism plays important roles in response to different salinity environments in spotted sea bass.

Salinity represents a critical environmental factor for fishes, and it can directly influence their survival. Transcriptomic analysis at the gene expression level has been extensively conducted to identify functional genes or pathways involved in salinity adaptation in numerous euryhaline fishes. However, the post-transcriptional regulation mechanism in response to salinity changes remains largely unknown. Alternative splicing (AS), the main mechanism accounting for the complexity of the transcriptome and proteome in eukaryotes, plays essential roles in determining organismal responses to environmental changes. In this study, RNA-Seq datasets were used to examine the AS profiles in spotted sea bass (Lateolabrax maculatus), a typical euryhaline fish species. The results showed that 8618 AS events were identified in spotted sea bass. Furthermore, a total of 501 and 162 differential alternative splicing (DAS) events were characterized in the gill and liver under low- and high-salinity environments, respectively. Based on GO enrichment results, DAS genes in both the gill and liver were commonly enriched in 8 GO terms, and their biological functions were implicated in many stages of gene expression regulation, including transcriptional regulation and post-transcriptional regulation. Sanger sequencing and qPCR validations provided additional evidence to ensure the accuracy and reliability of our bioinformatic results. This is the first comprehensive view of AS in response to salinity changes in fish species, providing insights into the post-regulatory molecular mechanisms of euryhaline fishes in salinity adaptation.

The DNA methylation status of MyoD and IGF-I genes are correlated with muscle growth during different developmental stages of Japanese flounder (Paralichthys olivaceus).

Many genes related to muscle growth modulate myoblast proliferation and differentiation and promote muscle hypertrophy. MyoD is a myogenic determinant that contributes to myoblast determination, and insulin-like growth factor 1 (IGF-I) interacts with MyoD to regulate muscle hypertrophy and muscle mass. In this study, we aimed to assess DNA methylation and mRNA expression patterns of MyoD and IGF-I during different developmental stages of Japanese flounder, and to examine the relationship between MyoD and IGF-I gene. DNA and RNA were extracted from muscles, and DNA methylation of MyoD and IGF-I promoter and exons was detected by bisulfite sequencing. The relative expression of MyoD and IGF-I was measured by quantitative polymerase chain reaction. IGF-I was measured by radioimmunoassay. Interestingly, the lowest expression of MyoD and IGF-I emerged at larva stage, and the mRNA expression was negatively associated with methylation. We hypothesized that many skeletal muscle were required to complete metamorphosis; thus, the expression levels of MyoD and IGF-I genes increased from larva stage and then decreased. The relative expression levels of MyoD and IGF-I exhibited similar patterns, suggesting that MyoD and IGF-I regulated muscle growth through combined effects. Changes in the concentrations of IGF-I hormone were similar to those of IGF-I gene expression. Our results the mechanism through which MyoD and IGF-I regulate muscle development and demonstrated that MyoD interacted with IGF-I to regulate muscle growth during different developmental stages.

DNA methylation levels and expression patterns of Smyd1a and Smyd1b genes during Metamorphosis of the Japanese Flounder (Paralichthys olivaceus).

Japanese flounder (Paralichthys olivaceus) undergoes metamorphosis by changing its body from the larval to the juvenile form, and this process involves muscle development. Smyd1, a histone methyltransferase, plays a role in the skeletal muscle. In the present study, the Smyd1a and Smyd1b expression patterns and their 5' UTR and exon 1 DNA methylation levels were analyzed during metamorphosis of the Japanese flounder. Sample were analyzed 21 days post-hatching (dph) (with no migration of right eye; M1stage), 28 dph (during migration of right eye; M2 stage), and 35 dph (after migration of right eye; M3 stage). The results show that Smyd1a expression was highest in the M2 stage and then decreased, whereas Smyd1b expression continued to rise during the three stages. Methylation levels of CpG sites at positions -2318 and -2217 of the Smyd1a P region (-2462 to -2181 region of the 5' UTR), and the CpG sites at positions -351, -330, -284, -190, and - 92 of the Smyd1b promoter, with both regions containing putative transcription factor binding sites, showed significant differences in the three stages (p < 0.05). Interestingly, the methylation levels of these CpG sites were negatively correlated with mRNA expression. We inferred that binding of the predicted transcription factors might be affected by methylation of the CpG sites and thus modulate gene expression. Taken together, our results suggest that DNA methylation in the Smyd1a and Smyd1b genes participates in the regulation of metamorphosis, and epigenetics may provide clues for further studies of the mechanisms of metamorphosis in the Japanese flounder.