Molecular Characterization and Expression Changes of the bcl2l13 Gene in Response to Hypoxia in Megalobrama amblycephala.

Hypoxia is a unique environmental stress, which not only reflects the insufficient oxygen supply of cells and tissues, but also occurs in various physiological and pathological environments. Mitophagy as a selective autophagy can recover and utilize damaged organelles and misfolded proteins to ensure normal cell functions and promote cell survival. Bcl2l13 (B-cell lymphoma-2 like 13) is reported to induce mitophagy as a functional mammalian homolog of Atg32. However, the function of the bcl2l13 gene is still unclear in fish. Here the sequence and structure of the bcl2l13 gene in Megalobrama amblycephala were identified and showed that bcl2l13 contained an open reading frame (ORF) of 1458 bp for encoding 485 aa. Amino acid sequence analysis indicated that Bcl2l13, as a typical anti-apoptotic protein of the Bcl2 family, contained four BH domains, one BHNo domain, and one TM domain. Further study showed that Bcl2l13 was mainly located in the mitochondria, while its localization was changed within the whole cell after the TM domain was deleted. Real-time PCR analysis revealed that bcl2l13 showed higher expression levels in early embryos. After hypoxia treatment, the mRNA levels of the bcl2l13 and autophagy-related genes were significantly up-regulated in most detected tissues, and the bcl2l13 transcription was regulated by Hif-1α mediated pathway. Additionally, the transcription activity of the bcl2l13 promoter was further analyzed using luciferase reporter assays and showed the highest activity in the promoter region from -475 to +111. These results indicated that bcl2l13 may play important roles in embryogenesis and hypoxia mediated autophagy in fish.

Janus kinase 1 in Megalobrama amblycephala: Identification, phylogenetic analysis and expression profiling after Aeromonas hydrophila infection.

Janus kinase 1 (JAK1), a member of the JAK family, plays an essential and non-redundant role in the mammalian immune system. However, the potential role of JAK1 in fish immune response remains largely unclear. In the present study, the JAK1 gene of Megalobrama amblycephala (MamJAK1) was identified and characterized. The open reading frame (ORF) of MamJAK1 was 3462 bp, encoding 1153 amino acids. MamJAK1 consists of four common domains of the JAK family, including B41, SH2, STyrKc (a pseudo kinase domain), and TyrKc (a kinase domain). Phylogenetic analysis showed that JAK1s are divided into two evolutionary clades, one containing fish JAK1s, and the other containing JAK1s from other vertebrates. The results of quantitative real-time PCR (qPCR) showed that in healthy M. amblycephala, MamJAK1 mRNA was highest expressed in blood, followed by spleen, intestine and mid-kidney, and lowly expressed in other tissues including gill, liver, head kidney, muscle, brain and heart. After Aeromonas hydrophila infection, the expression of MamJAK1 mRNA was significantly induced in four selected tissues including spleen, mid-kidney, liver and intestine, reaching a peak at 24 hpi (hour post infection) in spleen and mid-kidney, at 12 hpi in liver and at 4 hpi in intestine, and then the expression level was restricted to control levels at 72 or 120 hpi. In addition, the results of Western blot showed that the phosphorylation level of MamJAK1 protein in spleen and mid-kidney increased significantly after A. hydrophila infection, although MamJAK1 protein did not change obviously. Further, the JAK1 phosphorylation in Ctenopharyngodon idellus kidney (CIK) cells was found to be significantly induced by LPS stimulation and IL-6R over-expression. The results above suggest that MamJAK1 may play an essential role in the immune response against bacterial infection through the IL-6R mediated JAK1/STAT signaling pathway, which further deepen our understanding of JAK1 and provides a potential target for the treatment and prevention of bacterial diseases in teleost.

A Chromosome-Level Assembly of Blunt Snout Bream (Megalobrama amblycephala) Genome Reveals an Expansion of Olfactory Receptor Genes in Freshwater Fish.

The number of olfactory receptor genes (ORs), which are responsible for detecting diverse odor molecules varies extensively among mammals as a result of frequent gene gains and losses that contribute to olfactory specialization. However, how OR expansions/contractions in fish are influenced by habitat and feeding habit and which OR subfamilies are important in each ecological niche is unknown. Here, we report a major OR expansion in a freshwater herbivorous fish, Megalobrama amblycephala, using a highly contiguous, chromosome-level assembly. We evaluate the possible contribution of OR expansion to habitat and feeding specialization by comparing the OR repertoire in 28 phylogenetically and ecologically diverse teleosts. In total, we analyzed > 4,000 ORs including 3,253 intact, 122 truncated, and 913 pseudogenes. The number of intact ORs is highly variable ranging from 20 to 279. We estimate that the most recent common ancestor of Osteichthyes had 62 intact ORs, which declined in most lineages except the freshwater Otophysa clade that has a substantial expansion in subfamily ß and ε ORs. Across teleosts, we found a strong association between duplications of ß and ε ORs and freshwater habitat. Nearly, all ORs were expressed in the olfactory epithelium (OE) in three tested fish species. Specifically, all the expanded ß and ε ORs were highly expressed in OE of M. amblycephala. Together, we provide molecular and functional evidence for how OR repertoires in fish have undergone gain and loss with respect to ecological factors and highlight the role of ß and ε OR in freshwater adaptation.

The seahorse genome and the evolution of its specialized morphology.

Seahorses have a specialized morphology that includes a toothless tubular mouth, a body covered with bony plates, a male brood pouch, and the absence of caudal and pelvic fins. Here we report the sequencing and de novo assembly of the genome of the tiger tail seahorse, Hippocampus comes. Comparative genomic analysis identifies higher protein and nucleotide evolutionary rates in H. comes compared with other teleost fish genomes. We identified an astacin metalloprotease gene family that has undergone expansion and is highly expressed in the male brood pouch. We also find that the H. comes genome lacks enamel matrix protein-coding proline/glutamine-rich secretory calcium-binding phosphoprotein genes, which might have led to the loss of mineralized teeth. tbx4, a regulator of hindlimb development, is also not found in H. comes genome. Knockout of tbx4 in zebrafish showed a 'pelvic fin-loss' phenotype similar to that of seahorses.

High-temperature stress will put the thermo-sensitive teleost yellow catfish (Tachysurus fulvidraco) in danger through reducing reproductivity.

Recently, concerns for species that sex differentiation is influenced by temperature in the context of global warming have increased because disrupted operational sex ratios could threaten population maintenance. In contrast, little attention has been given to the reproductive ability of populations that experienced elevated temperatures. In this study, we demonstrated that high temperature (HT) would decrease population size via three different aspects of reproductive ability for the first time. We show that, in a thermo-sensitive teleost yellow catfish, a short period of HT (+3 °C) exposure during the critical period of sex differentiation leads to a different percentage of masculinization of XX genotypic females (1-23%) in wet-lab and natural water bodies. Combining the results of gonadal appearance, histology, sperm parameters, and fertilization rate, we found that XX pseudo-males induced by HT display significantly discounted fertility and reproductive performance compared to XY normal males. We demonstrate that the survival of the XY genotype is lower than XX genotype under environmental stress, including HT, hypoxia, and parasite infection, and the differential survival seems unrelated to male-biased sexual size dimorphism. The mathematical model predicts that the phenotypic female percent will be stabilized at 50% and the population will be sustainably maintained when masculinizing force is less than 0.5, while HT will put the population in danger when the masculinizing force exceeds 0.5. However, when we combine the real-world data of reproductive ability and mathematic model, our results suggest the population size decreases and the long-term survival of the studied species are threatened under the projected pace of increasing temperature. These findings will be useful for understanding the long-term effects of increasing temperature on sex ratio, reproduction and population maintenance in teleost.

Genome-wide analysis of intermuscular bone development reveals changes of key genes expression and signaling pathways in blunt snout bream (Megalobrama amblycephala).

Intermuscular bone (IB) is a hard-boned spicule exist in lower teleost, which brings a lot of detrimental effects on palatability and economic value of blunt snout bream (Megalobrama amblycephala). Masson trichrome staining for ossific IB indicated that some osteoblasts appeared at the edge of the bone matrix and a few osteocytes are present in the center of the mineralized bone matrix. By comparing the orthologous gene families of fish with IBs and without IBs, we screened the key signaling pathways associated with IB formation. Furthermore, the transcriptomic data demonstrated the functional importance of these gene families. The candidate genes involved in chondrocyte development were highly expressed in stage 1 compared with stage 2 and stage 3, suggesting that the development process of IB might mainly involve in intramembranous ossification. Our research reveals the molecular mechanism of IBs formation, and provides molecular evidence for the further study on intermuscular boneless stains.

Identification and Characterization of Novel circRNAs Involved in Muscle Growth of Blunt Snout Bream (Megalobrama amblycephala).

Circular RNAs (circRNAs), a novel class of endogenous RNAs, have been recognized to play important roles in the growth of animals. However, the regulatory mechanism of circRNAs on fish muscle growth is still unclear. In this study, we performed whole transcriptome analysis of skeletal muscles from two populations with different growth rates (fast-growing and slow-growing) of blunt snout bream (Megalobrama amblycephala), an important fish species for aquaculture. The selected circRNAs were validated by qPCR and Sanger sequencing. Pairs of circRNA-miRNA-mRNA networks were constructed with the predicted differentially expressed (DE) pairs, which revealed regulatory roles in muscle myogenesis and hypertrophy. As a result, a total of 445 circRNAs were identified, including 42 DE circRNAs between fast-growing (FG) and slow-growing (SG) groups. Many of these DE circRNAs were related with aminoglycan biosynthetic and metabolic processes, cytokinetic processes, and the adherens junction pathway. The functional prediction results showed that novel_circ_0001608 and novel_circ_0002886, competing to bind with dre-miR-153b-5p and dre-miR-124-6-5p, might act as competing endogenous RNAs (ceRNAs) to control MamblycephalaGene14755 (pik3r1) and MamblycephalaGene10444 (apip) level, respectively, thus playing an important regulatory role in muscle growth. Overall, these results will not only help us to further understand the novel RNA transcripts in M. amblycephala, but also provide new clues to investigate the potential mechanism of circRNAs regulating fish growth and muscle development.

Development of Teleost Intermuscular Bones Undergoing Intramembranous Ossification Based on Histological-Transcriptomic-Proteomic Data.

Intermuscular bones (IBs) specially exist in lower teleost fish and the molecular mechanism for its development remains to be clarified. In this study, different staining methods and comparative proteomics were conducted to investigate the histological structure and proteome of IB development in Megalobrama amblycephala, including four key IB developmental stages (S1-IBs have not emerged in the tail part; S2-several small IBs started to ossify in the tail part; S3-IBs appeared rapidly; S4-all the IBs appeared with mature morphology). Alcian blue and alizarin red S stained results indicated that IBs were gradually formed from S2 to S4, undergoing intramembranous ossification without a cartilaginous phase. A total of 3368 proteins were identified by using the isobaric tags for relative and absolute quantitation (iTRAQ) approach. Functional annotation showed that proteins which were differentially expressed among stages were involved in calcium, MAPK, Wnt, TGF-ß, and osteoclast pathways which played a critical role in bone formation and differentiation. Three proteins (collagen9α1, stat1, tnc) associated with chondrocytes did not exhibit significant changes through S2 to S4; however, proteins (entpd5, casq1a, pvalb, anxa2a, anxa5) which associated with osteoblasts and bone formation and differentiation showed significantly a higher expression level from S1 to S2, as well as to S3 and S4. These further demonstrated that development of IBs did not go through a cartilaginous phase. The inhibitors of TGF-ß and Wnt pathways were tested on zebrafish (sp7/eGFP) and the results indicated that both inhibitors significantly delayed IB development. This study provides a comprehensive understanding of the IB ossification pattern, which will help further elucidate the molecular mechanisms for IB development in teleosts.

Comparative expression analysis of let-7 microRNAs during ovary development in Megalobrama amblycephala.

As a critical regulator of gene expression, let-7 family miRNAs have been reported to be involved in multiple physiological processes. In this study, in order to elucidate the putative regulatory effect of let-7 miRNAs during fish gonadal development and to identify which member is crucial for this regulation, the expression of ten members including let-7a/b/c/d/e/f/g/h/i/j were quantified in ovary, pituitary, and brain tissues during the different ovarian developmental stages of blunt snout bream Megalobrama amblycephala. According to the data from analysis of expression patterns, let-7a showed the highest expression value in almost all the tested ovaries, pituitaries, and brains, with let-7b and let-7d moderately expressed, following by other let-7 miRNAs. In terms of the differential expression levels of ten let-7 miRNAs at each developmental stage, the results showed that let-7a/b/d/f/h expression gradually increased during the ovary development from stage I to V and dropped significantly at the phase VI in ovary tissues. However, the expression of let-7a/b/e/f in pituitary increased during the ovary development from stage I to IV and declined at stage V. Among all the let-7 miRNAs, let-7a/b/d had the highest expression and their expression patterns were consistent with the gonad development of M. amblycephala. Furthermore, the mostly predicted target genes of let-7 miRNAs are significantly enriched in signaling pathways closely related to gonadal development through KEGG enrichment analysis. These results indicate that let-7 miRNA members, especially let-7a/b/d, may play important roles in the regulation of ovary development in M. amblycephala through negatively regulating expression of their target genes.

Is the Nutritional Value of Fish Fillet Related to Fish Maturation or Fish Age? Integrated Analysis of Transcriptomics and Metabolomics in Blunt Snout Bream (Megalobrama amblycephala).

BACKGROUND/AIMS: Fish is a protein-rich food and is increasingly favored by consumers. It has been well recognized that the flesh composition of fish is closely related to its maturation and growth stage, but few studies have explored these differences. Additionally, hormone residues in fish after artificial induction of reproduction also attract consumer concern. In this study, we attempt to address these concerns by using a combination of transcriptomics and metabolomics analyses to identify key regulated pathways, genes, and metabolites that may affect the flesh nutrition of one typical aquaculture species in China, blunt snout bream (Megalobrama amblycephala). METHODS: The four groups of fish were used for transcriptomics and metabolomics analyses, including one-year-old immature (group I), two-year-old immature (group II), two-year-old mature (group III) and successfully spawned (group IV) female M. amblycephala after artificial induction of reproduction. RESULTS: We identified a total of 1460 differential compounds and 1107 differentially expressed unigenes in muscle among the different groups. Differential metabolites related to fish age (group II vs group I, group III vs group I) were largely enriched in "Glycerophospholipid metabolism", "Linoleic acid metabolism", "α-Linolenic acid metabolism", and "Biosynthesis of unsaturated fatty acids". Between these two pairwise comparisons, metabolites that are beneficial to human health, such as docosapentaenoic acid, α-Linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid were found to be significantly decreased in two-year-old (group II, group III) compared with one-year-old (group I) M. amblycephala. Only one differential metabolite related to fish maturation, a triglyceride, was detected between groups III and II. Transcriptomics data showed that differently expressed genes (between group III vs group II, group III vs group I) related to maturation were highly enriched in "Cell adhesion molecules (CAMs)", "Sphingolipid metabolism" and "Phagosome". DEGs (between group II vs group I, group III vs group I) relating to fish age were enriched in the "cGMP-PKG signaling pathway", "FoxO signaling pathway", and "AMPK signaling pathway". The gene-metabolite interaction network showed pivotal genes, including fumarate hydratase and GNPAT, which played a major role in the regulation of glycerphospholipid metabolism. The nutritional components were also measured, which verified the metabolomics results. Moreover, the metabolomics results showed that after 24 hours of artificial hormone injection, the drug was completely metabolized. CONCLUSION: Integrated analysis demonstrated that the nutrition value of fish fillet was much more related to fish age compared with maturation status in M. amblycephala females.

Comparative proteomics analysis of teleost intermuscular bones and ribs provides insight into their development.

BACKGROUND: Intermuscular bones (IBs) and ribs both are a part of skeletal system in teleosts, but with different developing process. The chemical composition of fish IBs and ribs as well as the underlying mechanism about their development have not been investigated. In the present study, histological structures showed that one bone cavity containing osteoclasts were existed in ribs, but not in IBs of Megalobrama amblycephala. We constructed the first proteomics map for fish bones including IBs and ribs, and identified the differentially expressed proteins between IBs and ribs through iTRAQ LC-MS/MS proteomic analysis. RESULTS: The proteins extracted from IBs and ribs at 1- to 2-year old M. amblycephala were quantified 2,342 proteins, with 1,451 proteins annotated with GO annotation in biological processes, molecular function and cellular component. A number of bone related proteins as well as pathways were identified in the study. A total of 93 and 154 differently expressed proteins were identified in comparison groups of 1-IB-vs-1-Rib and 2-IB-vs-2-Rib, which indicated the obvious differences of chemical composition between these two bone tissues. The two proteins (vitronectin b precursor and matrix metalloproteinase-2) related to osteoclasts differentiation were significantly up-regulated in ribs compared with IBs (P < 0.05), which was in accordance with the results from histological structures. In comparison groups of 1-IB-vs-2-IB and 1-Rib-vs-2-Rib, 33 and 51 differently expressed proteins were identified and the function annotation results showed that these proteins were involved in regulating bone development and differentiation. Subsequently, 11 and 13 candidate proteins in comparison group of 1-IB-vs-1-Rib and 1-IB-vs-2-IB related to bone development were validated by MRM assays. CONCLUSIONS: Our present study suggested the different key proteins involved in the composition of fish ribs and IBs as well as their growth development. These findings could provide important clues towards further understanding of fish skeletal system and the roles of proteins playing in regulating diverse biological processes in fish.

Evolution of Fish Let-7 MicroRNAs and Their Expression Correlated to Growth Development in Blunt Snout Bream.

The lethal-7 (let-7) miRNA, known as one of the first founding miRNAs, is present in multiple copies in a genome and has diverse functions in animals. In this study, comparative genomic analysis of let-7 miRNAs members in fish species indicated that let-7 miRNA is a sequence conserved family in fish, while different species have the variable gene copy numbers. Among the ten members including let-7a/b/c/d/e/f/g/h/i/j, the let-7a precursor sequence was more similar to ancestral sequences, whereas other let-7 miRNA members were separate from the late differentiation of let-7a. The mostly predicted target genes of let-7 miRNAs are involved in biological process, especially developmental process and growth through Gene Ontology (GO) enrichment analysis. In order to identify the possible different functions of these ten miRNAs in fish growth development, their expression levels were quantified in adult males and females of Megalobrama amblycephala, as well as in 3-, 6-, and 12-months-old individuals with relatively slow- and fast-growth rates. These ten miRNAs had similar tissue expression patterns between males and females, with higher expression levels in the brain and pituitary than that in other tissues (p < 0.05). Among these miRNAs, the relative expression level of let-7a was the highest among almost all the tested tissues, followed by let-7b, let-7d and let-7c/e/f/g/h/i/j. As to the groups with different growth rates, the expression levels of let-7 miRNAs in pituitary and brain from the slow-growth group were always significantly higher than that in the fast-growth group (p < 0.05). These results suggest that let-7 miRNA members could play an important role in the regulation of growth development in M. amblycephala through negatively regulating expression of their target genes.

Molecular mechanism of endocrine system impairment by 17α-methyltestosterone in gynogenic Pengze crucian carp offspring.

The effects of synthetic androgen 17α-methyltestosterone (MT) on endocrine impairment were examined in crucian carp. Immature 7-month old mono-female Pengze crucian carp (Pcc) F2 offspring were exposed to 50 and 100 µg/L of MT (week 2, 4, and 8). Gonadosomatic index, hepatosomatic index and intestine weight altered considerably and oocyte development was repressed. In the treatment groups, ovarian 11-ketotestosterone decreased, whereas 17ß-estradiol and testosterone increased, and ovarian aromatase activities increased at week 4. However, in the brain tissue, those values significantly decreased. Quantitative RT-PCR analysis demonstrated changes in steroid receptor genes and upregulation of steroidogenic genes (Pcc-3bhsd, Pcc-11bhsd2 Pcc-cyp11a1), while the other three steroidogenic genes (Pcc-cyp17a1, Pcc-cyp19a1a and Pcc-star) decreased from week 4 to week 8. Ovarian, hepatic Pcc-vtg B and vitellogenin concentration increased in both 50 and 100 µg/L of MT exposure groups. This study adds further information regarding the effects of androgens on the development of previtellogenic oocytes, which suggests that MT could directly target estrogen signaling pathway, or indirectly affect steroidogenesis and vitellogenesis.

Transcriptome analysis and microsatellite discovery in the blunt snout bream (Megalobrama amblycephala) after challenge with Aeromonas hydrophila.

The blunt snout bream, Megalobrama amblycephala, is a herbivorous freshwater fish species native to China and a major aquaculture species in Chinese freshwater polyculture systems. In recent years, the bacterium Aeromonas hydrophila has been reported to be its pathogen causing great losses of farmed fish. To understand the immune response of the blunt snout bream to A. hydrophila infection, we used the Solexa/Illumina technology to analyze the transcriptomic profile after artificial bacterial infection. Two nonnormalized cDNA libraries were synthesized from tissues collected from control blunt snout bream or those injected with A. hydrophila. After assembly, 155,052 unigenes (average length 692.8 bp) were isolated. All unigenes were annotated using BLASTX relative to several public databases: the National Center for Biotechnology Information nonreduntant (Nr) database, SwissProt, Eukaryotic Orthologous Groups of proteins (KOG), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Ontology (GO). The sequence similarity (86%) of the assembled unigenes was to zebrafish based on the Nr database. A number of unigenes (n = 30,482) were assigned to three GO categories: biological processes (25,242 unigenes), molecular functions (26,096 unigenes), and cellular components (22,778 unigenes). 20,909 unigenes were classified into 25 KOG categories and 28,744 unigenes were assigned into 315 specific signaling pathways. In total, 238 significantly differentially expressed unigenes (mapped to 125 genes) were identified: 101 upregulated genes and 24 downregulated genes. Another 303 unigenes were mapped to unknown or novel genes. Among the known expressed genes identified, 53 were immune-related genes and were distributed in 71 signaling pathways. The expression patterns of selected up- and downregulated genes from the control and injected groups were determined with reverse transcription-quantitative PCR (RT-qPCR). Microsatellites (n = 10,877), including di-to pentanucleotide repeat motifs, were also identified in the blunt snout bream transcriptome profiles. This study extends our understanding of the immune defense mechanisms of the blunt snout bream against A. hydrophila and provides useful data for further studies of the immunogenetics of this species.

Molecular characterisation and prevalence of a new genotype of Cyprinid herpesvirus 2 in mainland China.

Cyprinid herpesvirus 2 (CyHV-2, species Cyprinid herpesvirus 2) has been confirmed as a causative agent of the acute haematopoietic necrosis disease outbreak in farmed goldfish (Carassius auratus L.) and gibel carp (Carassius auratus gibelio Bloch). In this study, we present the genomic characteristics of a variant CyHV-2 strain (SY-C1) isolated from farmed gibel carp in mainland China and its comparative genomics analysis with the CyHV-2 reference strain ST-J1. Overall, the full-length genome of SY-C1 shares 98.8% homology with that of ST-J1. Sequence comparisons between SY-C1 and ST-J1 indicate that the variations include single-nucleotide mutations, insertions, deletions, and rearrangements, which suggested that SY-C1 is different from ST-J1 and represents a new genotype. Therefore, we propose that the identified CyHV-2 can be divided into 2 different genotypes and be named China genotype (C genotype) and Japan genotype (J genotype) according to their isolation loci. Furthermore, epidemiological surveys indicate that the dominant genotype of CyHV-2 circulating in mainland China is closer to the China genotype than the Japan genotype.

Leptin Genes in Blunt Snout Bream: Cloning, Phylogeny and Expression Correlated to Gonads Development.

To investigate the leptin related genes expression patterns and their possible function during the gonadal development in fish, the cDNA and genomic sequences of leptin, leptin receptor (leptinR), and leptin receptor overlapping transcript like-1 (leprotl1) were cloned and their expression levels were quantified in the different gonadal development stages of Megalobrama amblycephala. The results showed that the full length cDNA sequences of leptin, leptinR and leprotl1 were 953, 3432 and 1676 bp, coding 168, 1082, and 131 amino acid polypeptides, and the genomic sequences were 1836, 28,528 and 5480 bp, which respectively had 3, 15 and 4 exons, respectively. The phylogenetic analysis revealed that three genes were relatively conserved in fish species. Quantitative real-time PCR results showed that the three genes were ubiquitously expressed in all examined tissues during the different gonadal development stages. The leptin and leptinR took part in the onset of puberty, especially in female M. amblycephala, by increasing the expression levels in brain during the stage I to III of ovary. The expression levels of leptin and leptinR had significant differences between male and female in hypothalamic-pituitary-gonadal (HPG) axis tissues (p < 0.05). The leptinR had the same variation tendency with leptin, but the opposite changes of expression levels were found in leprotl1, which may resist the expression of leptinR for inhibiting the function of leptin in target organ. These findings revealed details about the possible role of these genes in regulating gonadal maturation in fish species.

Identification of MicroRNA for Intermuscular Bone Development in Blunt Snout Bream (Megalobrama amblycephala).

Intermuscular bone (IB), which occurs only in the myosepta of the lower teleosts, is attracting more attention of researchers due to its particular development and lack of genetic information. MicroRNAs (miRNAs) are emerging as important regulators for biological processes. In the present study, miRNAs from IBs and connective tissue (CT; encircled IBs) from six-month-old Megalobrama amblycephala were characterized and compared. The results revealed the sequences and expression levels of 218 known miRNA genes (belonging to 97 families). Of these miRNAs, 44 known microRNA sequences exhibited significant expression differences between the two libraries, with 24 and 20 differentially-expressed miRNAs exhibiting higher expression in the CT and IBs libraries, respectively. The expressions of 11 miRNAs were selected to validate in nine tissues. Among the high-ranked predicted gene targets, differentiation, cell cycle, metabolism, signal transduction and transcriptional regulation were implicated. The pathway analysis of differentially-expressed miRNAs indicated that they were abundantly involved in regulating the development and differentiation of IBs and CT. This study characterized the miRNA for IBs of teleosts for the first time, which provides an opportunity for further understanding of miRNA function in the regulation of IB development.

The Regulatory Role of Myomaker in the Muscle Growth of the Chinese Perch (Siniperca chuatsi).

The fusion of myoblasts is a crucial stage in the growth and development of skeletal muscle. Myomaker is an important myoblast fusion factor that plays a crucial role in regulating myoblast fusion. However, the function of Myomaker in economic fish during posthatching has been poorly studied. In this study, we found that the expression of Myomaker in the fast muscle of Chinese perch (Siniperca chuatsi) was higher than that in other tissues. To determine the function of Myomaker in fast muscle, Myomaker-siRNA was used to knockdown Myomaker in Chinese perch and the effect on muscle growth was determined. The results showed that the growth of Chinese perch was significantly decreased in the Myomaker-siRNA group. Furthermore, both the diameter of muscle fibers and the number of nuclei in single muscle fibers were significantly reduced in the Myomaker-siRNA group, whereas there was no significant difference in the number of BrdU-positive cells (proliferating cells) between the control and the Myomaker-siRNA groups. Together, these findings indicate that Myomaker may regulate growth of fast muscle in Chinese perch juveniles by promoting myoblast fusion rather than proliferation.

Chromosome-level genome assembly of the yellow-cheek carp Elopichthys bambusa.

Yellow-cheek carp (Elopichthys bambusa) is a typical large and ferocious carnivorous fish endemic to East Asia, with high growth rate, nutritional value and economic value. In this study, a chromosome-level genome of yellow-cheek carp was generated by combining PacBio reads, Illumina reads and Hi-C data. The genome size is 827.63 Mb with a scaffold N50 size of 33.65 Mb, and 99.51% (823.61 Mb) of the assembled sequences were anchored to 24 pseudo-chromosomes. The genome is predicted to contain 24,153 protein-coding genes, with 95.54% having functional annotations. Repeat elements account for approximately 55.17% of the genomic landscape. The completeness of yellow-cheek carp genome assembly is highlighted by a BUSCO score of 98.4%. This genome will help us understand the genetic diversity of yellow-cheek carp and facilitate its conservation planning.

Muscle Fiber Characteristics and Transcriptome Analysis in Slow- and Fast-Growing Megalobrama amblycephala.

Growth is an important trait in aquaculture that is influenced by various factors, among which genetic regulation plays a crucial role. Megalobrama amblycephala, one of the most important freshwater species in China, exhibits wide variations in body mass among individuals of the same age within the same pool. But the molecular mechanisms underlying wide variation in body mass remain unclear. Here, we performed muscle histological and transcriptome analysis of muscle tissues from Fast-Growing (FG) and Slow-Growing (SG) M. amblycephala at the age of 4 months old (4 mo) and 10 months old (10 mo) to elucidate its muscle development and growth mechanism. The muscle histological analysis showed smaller diameter and higher total number of muscle fibers in FG compared to SG at 4 mo, while larger diameter and total number of muscle fibers were detected in FG at 10 mo. The transcriptome analysis of muscle tissue detected 1171 differentially expressed genes (DEGs) between FG and SG at 4 mo, and 718 DEGs between FG and SG at 10 mo. Furthermore, 44 DEGs were consistently up-regulated in FG at both 4 mo and 10 mo. Up-regulated DEGs in FG at 4 mo were mainly enriched in the pathways related to cell proliferation, while down-regulated DEGs were significantly enriched in cell fusion and muscle contraction. Up-regulated DEGs in FG at 10 mo were mainly enriched in the pathways related to cell proliferation and protein synthesis. Therefore, these results provide novel insights into the molecular mechanism of M. amblycephala muscle growth at different stages, and will be of great guiding significance to promote the fast growth of M. amblycephala.