Combining genome-wide association study and transcriptome analysis to identify molecular markers and genetic basis of population-asynchronous ovarian development in Coilia nasus.

Coilia nasus, a migratory fish species found in the middle and lower reaches of the Yangtze River and along offshore areas of China, possesses considerable aquacultural and economic potential. However, the species faces challenges due to significant variation in the gonadal development rate among females, resulting in inconsistent ovarian maturation times at the population level, an extended reproductive period, and limitations on fish growth rate due to ovarian prematurity. In the present study, we combined genome-wide association study (GWAS) and comparative transcriptome analysis to investigate the potential single nucleotide polymorphisms (SNPs) and candidate genes associated with population-asynchronous ovarian development in C. nasus. Genotyping of the female population based on whole-genome resequencing yielded 2 120 695 high-quality SNPs, 39 of which were suggestively associated with ovarian development. Of note, a significant SNP peak on LG21 containing 30 suggestively associated SNPs was identified, with cpne5a determined as the causal gene of the peak. Therefore, single-marker and haplotype association analyses were performed on cpne5a, revealing four genetic markers ( P<0.05) and seven haplotypes (r 2>0.9) significantly associated with the phenotype. Comparative transcriptome analysis of precociously and normally maturing individuals screened out 29 and 426 overlapping differentially expressed genes in the brain and ovary, respectively, between individuals of different body sizes. Integrating the GWAS and transcriptome analysis results, this study identified genes and pathways related to hypothalamic-pituitary-gonadal axis hormone secretion, extracellular matrix, angiogenesis, and gap junctions involved in population-asynchronous ovarian development. The insights gained from this study provide a basis for a deeper understanding of the molecular mechanisms underlying ovarian development in fish and may facilitate the genetic breeding of C. nasus strains exhibiting population-synchronous ovarian development in the future.

Comparative transcriptome profiles of four sexually size dimorphic fish.

Sexual size dimorphism is widespread in fish species. Although sex growth differences in multiple species have been studied successively, the commonalities of regulatory mechanisms across sexually dimorphic species are unknown. In this study, we performed RNA-seq analysis of four representative fish (loach, half-smooth tongue sole, yellow catfish, and Nile tilapia) with significant growth differences between females and males. Clean reads were identified from four fish species, ranging from 45,718,052 to 57,733,120. Following comparison transcriptome analysis, there were 1,132 and 1,108, 1,290 and 1,102, 4,732 and 4,266, 748 and 192 differentially expressed genes (DEGs) in the brain and muscle of loach, half-smooth tongue sole, yellow catfish, and Nile tilapia, respectively. Furthermore, the expression levels were validated by quantitative real-time PCR (qRT-PCR). Comparative transcriptome profiles of four fish described here will provide fundamental information for further studies on the commonalities of sexually size dimorphic fish in regulating growth differences between females and males.

Single-cell transcriptomes and runx2b-/- mutants reveal the genetic signatures of intermuscular bone formation in zebrafish.

Intermuscular bones (IBs) are mineralized spicules, present in the myosepta of many, but not all, teleost species. IBs are often small and sharp, and they consequently limit how the fish can be processed; the IBs may cause injury or trauma if lodged in consumers' throats or mouths, and therefore affect the appeal of the fish to many consumers. The development of IBs in teleosts is still not fully understood and the molecular basis of IB development remains to be established. Here, the characteristics of IB tissue are evaluated based on single-cell transcriptomics in wild-type zebrafish. The analysis defined 18 distinct cell types. Differentiation trajectories showed that IBs are derived from tendons and that a core tendon-osteoblast cell lineage is related to IB formation. In particular, the functions of 10 candidate genes were evaluated via CRISPR-Cas9 mutants. Among those, runx2b-/- mutants completely lost IBs, while swimming performance, growth and bone mineral density were not significantly different from runx2b+/+ zebrafish. Comparative single-cell RNA sequencing (scRNA-seq) analysis in runx2b-/- and runx2b+/+ zebrafish revealed the role of osteoblasts in IB formation. In addition, differentially expressed genes were enriched in the transforming growth factor ß/bone morphogenetic protein (TGF-ß/BMP) pathway after runx2b deletion. This study provides evidence for the crucial role of runx2b regulation in IB formation. Genetic breeding can target runx2b regulation and generate strains of commercial fish species without IBs, which can improve the safe consumption and economic value of many farmed fish species.

Functional differentiation of bmp2a and bmp2b genes in zebrafish.

Bone morphogenetic protein 2 plays an important role in the regulation of osteoblast proliferation and differentiation. Phylogenetic analysis showed that the bmp2 ortholog evolved from the same ancestral gene family in vertebrates and was duplicated in teleost, which were named bmp2a and bmp2b. The results of whole-mount in situ hybridization showed that the expression locations of bmp2a and bmp2b in zebrafish were different in different periods (24 hpf, 48 hpf, 72 hpf), which revealed potential functional differentiation between bmp2a and bmp2b. Phenotypic analysis showed that bmp2a mutations caused partial rib and vertebral deformities in zebrafish, while bmp2b-/- embryos died massively after 12 hpf due to abnormal somite formation. We further explored the expression pattern changes of genes (bmp2a, bmp2b, smad1, fgf4, runx2b, alp) related to skeletal development at different developmental stages (20 dpf, 60 dpf, 90 dpf) in wild-type and bmp2a-/- zebrafish. The results showed that the expression of runx2b in bmp2a-/- was significantly downregulated at three stages and the expression of other genes were significantly downregulated at 90 dpf compared with wild-type zebrafish. The study revealed functional differentiation of bmp2a and bmp2b in zebrafish embryonic and skeletal development.

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.

Functional Differentiation of BMP7 Genes in Zebrafish: bmp7a for Dorsal-Ventral Pattern and bmp7b for Melanin Synthesis and Eye Development.

Bone morphogenetic protein 7 (BMP7) belongs to the transforming growth factor ß (TGF-ß) family, which not only induces cartilage and bone formation, but also regulates eye development and melanoma tumorigenesis in mammals. In teleosts, BMP7 differentiates into two subtypes, bmp7a and bmp7b, which have clearly differentiated structures. To fully understand the functional differentiation of bmp7a and bmp7b in fish species, we successfully constructed bmp7a and bmp7b gene deletion mutants in zebrafish using CRISPR/Cas9-mediated gene editing technology. Our results showed that bmp7a mutation caused abnormal development of the embryo's dorsal-ventral pattern that led to death; bmp7b mutation induced growth inhibition and increased melanin production in the skin and eye of mutants. Histological analysis revealed that melanin in the retina of the eyes in bmp7b mutants increased, and behavioral observation showed that the vision and sensitivity to food of the mutants were reduced. Transcriptome analysis of the skin and eye tissues showed that the expression changes of wnt7ba and gna14 in bmp7b mutants might promote the increase of melanin. Additionally, the eye transcriptome analysis indicated that changes in the structure of the eyes in bmp7b mutants led to defects in phototransduction, and seven DEGs (rgs9a, rgs9b, rcvrn2, guca1d, grk1b, opn1mw4, and gc2) were identified as key candidate genes that affected the photonic response of the eyes. The study revealed the functional differentiation of bmp7a and bmp7b in teleosts and the first report about the inhibitory effect of bmp7b on melanogenesis may provide useful information for the future research on human melanoma-related diseases.

Comparison of Myosepta Development and Transcriptome Profiling between Blunt Snout Bream with and Tilapia without Intermuscular Bones.

Intermuscular bones (IBs) are small spicule-like bones located in the myosepta of basal teleosts, which negatively affect the edibleness and economic value of fish. Blunt snout bream (Megalobrama amblycephala, with epineural and epipleural IBs) and tilapia (Oreochromis niloticus, without epineural and epipleural IBs) are two major aquaculture species and ideal models for studying the formation mechanisms of fish IBs. Here, we compared myosepta development between M. amblycephala and O. niloticus, based on histological analysis, transcriptome profiling, and expression analysis of bone-related genes. The histological results showed that dye condensation began to appear in the myosepta 20 days post hatching (dph) in M. amblycephala, and IBs could be clearly observed 50 dph in the myosepta, based on different staining methods. However, in O. niloticus, dye condensation was not observed in the myosepta from 10 to 60 dph. Differentially expressed genes (DEGs) at different developmental stages were screened by comparing the transcriptomes of M. amblycephala and O. niloticus, and KEGG analysis demonstrated that these DEGs were enriched in many bone-related pathways, such as focal adhesion, calcium, and Wnt signaling pathways. Quantitative PCR was performed to further compare the expression levels of some bone-related genes (scxa, scxb, runx2a, runx2b, bgp, sp7, col1a2, entpd5a, entpd5b, phex, alpl, and fgf23). All the tested genes (except for alpl) exhibited higher expression levels in M. amblycephala than in O. niloticus. A comparison of the dorsal and abdominal muscle tissues between the two species also revealed significant expression differences for most of the tested genes. The results suggest that scxa, scxb, runx2a, runx2b, entpd5a, col1a2, and bgp may play important roles in IB development. Our findings provide some insights into the molecular mechanisms of IB formation, as well as clues for further functional analysis of the identified genes to better understand the development of IBs.

Comparative Transcriptome Analysis Revealed Genes Involved in Sexual and Polyploid Growth Dimorphisms in Loach (Misgurnus anguillicaudatus).

Sexual and polyploidy size dimorphisms are widespread phenomena in fish, but the molecular mechanisms remain unclear. Loach (Misgurnus anguillicaudatus) displays both sexual and polyploid growth dimorphism phenomena, and are therefore ideal models to study these two phenomena. In this study, RNA-seq was used for the first time to explore the differentially expressed genes (DEGs) between both sexes of diploid and tetraploid loaches in four tissues (brain, gonad, liver, and muscle). Results showed that 21,003, 17, and 1 DEGs were identified in gonad, liver, and muscle tissues, respectively, between females and males in both diploids and tetraploids. Regarding the ploidy levels, 4956, 1496, 2187, and 1726 DEGs were identified in the brain, gonad, liver, and muscle tissues, respectively, between tetraploids and diploids of the same sex. When both sexual and polyploid size dimorphisms were considered simultaneously in the four tissues, only 424 DEGs were found in the gonads, indicating that these gonadal DEGs may play an important regulatory role in regulating sexual and polyploid size dimorphisms. Regardless of the sex or ploidy comparison, the significant DEGs involved in glycolysis/gluconeogenesis and oxidative phosphorylation pathways were upregulated in faster-growing individuals, while steroid hormone biosynthesis-related genes and fatty acid degradation and elongation-related genes were downregulated. This suggests that fast-growing loaches (tetraploids, females) have higher energy metabolism levels and lower steroid hormone synthesis and fatty acid degradation abilities than slow-growing loaches (diploids, males). Our findings provide an archive for future systematic research on fish sexual and polyploid dimorphisms.

A comparative genomic database of skeletogenesis genes: from fish to mammals.

Skeletogenesis is a complex process that requires a rigorous control at multiple levels during osteogenesis, such as signaling pathways and transcription factors. The skeleton among vertebrates is a highly conserved organ system, but teleost fish and mammals have evolved unique traits or have lost particular skeletal elements in each lineage. In present study, we constructed a skeletogenesis database containing 4101, 3715, 2996, 3300, 3719 and 3737 genes in Danio rerio, Oryzias latipes, Gallus gallus, Xenopus tropicalis, Mus musculus and Homo sapiens genome, respectively. Then, we found over 55% of the genes are conserved in the six species. Notably, there are 181 specific-genes in the human genome without orthologues in the other five genomes, such as the ZNF family (ZNF100, ZNF101, ZNF14, CALML6, CCL4L2, ZIM2, HSPA6, etc); and 31 genes are identified explicitly in fish species, which are mainly involved in TGF-beta, Wnt, MAPK, Calcium signaling pathways, such as bmp16, bmpr2a, eif4e1c, wnt2ba, etc. Particularly, there are 20 zebrafish-specific genes (calm3a, si:dkey-25li10, drd1a, drd7, etc) and one medaka-specific gene (c-myc17) that may alter skeletogenesis formation in the corresponding species. The database provides the new systematic genomic insights into skeletal development from teleosts to mammals, which may help to explain some of the complexities of skeletal phenotypes among different vertebrates and provide a reference for the treatment of skeletal diseases as well as for applications in the aquaculture industry.

Screening of Biomarkers Related to Ovarian Maturation and Spawning in Blunt Snout Bream (Megalobrama amblycephala) Based on Metabolomics and Transcriptomics.

In fish breeding practices, gamete maturity of females is vital to reproductive success. For some species, it is possible to estimate the female maturation status based on abdomen observation, but quite difficult for some species which mature at big size. To screen out the potential biomarker in fish blood relating to female maturation, we employed the approach integrating the UPLC-MS/MS and RNA-seq techniques to investigate the metabolites and genes reflecting the sexual maturation and spawning of female blunt snout bream Megalobrama amblycephala. The study included four groups, 1-year-old immature female individuals, 2-year-old immature female individuals, 2-year-old sexually mature female individuals, and 2-year-old sexually mature female individuals after 24 h of successful spawning. The upregulated metabolites in mature females were involved in "steroid hormone biosynthesis," "metabolic pathways," "glycerophospholipid metabolism," etc. compared with those of immature individuals. As the key intermediate of steroid hormone biosynthesis, 17α-hydroxypregnenolone exhibited the highest level in 2-year-old mature females than in the immature females. Meanwhile, the metabolites (i.e., dodecanoic acid and myristic acid) participating in fatty acid synthesis exhibited much lower levels in the females after spawning than those before spawning. In addition to the metabolites, the genes involved in ovarian steroidogenesis were significantly upregulated in the 2-year-old immature females compared to the 1-year-old immature females, indicating that the ovarian steroidogenesis plays important roles in ovarian development of M. amblycephala at the early stages. The significant upregulation of genes (i.e., itpr1, camk2, and mekk2) involved in the "GnRH signaling pathway" was observed in the mature females compared with the immature females, which indicated that the estrogen levels increased after female maturation in M. amblycephala. Moreover, many genes (e.g., gck, creb1, tf2-9, ryr2, asgr1, and creb1) regulating insulin secretion and thyroid hormone synthesis were significantly downregulated after female spawning. The dynamics of gene expression and metabolites observed in this study provide novel cues for guiding fish practical artificial reproduction.

Genome-Wide Integrated Analysis Revealed Functions of lncRNA-miRNA-mRNA Interaction in Growth of Intermuscular Bones in Megalobrama amblycephala.

Intermuscular bone (IB) occurs in the myosepta of teleosts. Its existence has an adverse influence on the edible and economic value of fish, especially for aquaculture species belonging to Cypriniformes. The growth mechanism of IBs is quite lacking. In this study, we firstly used single molecular real-time sequencing (SMRT) technology to improve the draft genome annotation and full characterization of the transcriptome for one typical aquaculture species, blunt snout bream (Megalobrama amblycephala). The long non-coding RNA (lncRNA), microRNA (miRNA), and messenger RNA (mRNA) expression profiles in two IB growth stages (1 and 3 years old) were compared through transcriptome and degradome analyses. A total of 126 miRNAs, 403 mRNAs, and 353 lncRNAs were found to be differentially expressed between the two stages. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the significantly upregulated map2k6 and cytc in the MAPK/p53 signaling pathway and the significantly downregulated lama3 and thbs4b in the extracellular matrix (ECM)-receptor pathway may play a key regulatory role in IB growth. Bioinformatics analysis subsequently revealed 14 competing endogenous RNA (ceRNA) pairs related to the growth of IBs, consisting of 10 lncRNAs, 7 miRNAs, and 10 mRNAs. Of these, dre-miR-24b-3p and dre-miR-193b-3p are core regulatory factors interacting with four lncRNAs and three mRNAs, the interaction mechanism of which was also revealed by subsequent experiments at the cellular level. In conclusion, our data showed that IBs had higher activity of cell apoptosis and lower mineralization activity in IB_III compared to IB_I via interaction of MAPK/p53 and ECM-receptor signaling pathways. The downregulated zip1 interacted with miR-24a-3p and lnc017705, decreased osteoblast differentiation and Ca2+ deposition in the IB_III stage. Our identified functional mRNAs, lncRNAs, and miRNAs provide a data basis for in-depth elucidation of the growth mechanism of teleost IB.

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.

Characterization and spatiotemporal expression analysis of nine bone morphogenetic protein family genes during intermuscular bone development in blunt snout bream.

Intermuscular bones (IBs) only exist in the myosepta of lower teleosts and its molecular mechanism remains to be clarified. Bone morphogenetic proteins (BMPs) have been demonstrated to be involved in various physiological processes, including bone and cartilage formation. In this study, we firstly obtained and characterized nine bmp genes for Megalobrama amblycephala, which belongs to Cyprinidae and have a certain amount of IBs. Sequence alignment and phylogenetic analysis both documented that the mature proteins of M. amblycephala bmp genes were highly conserved with other corresponding homologs, respectively, indicating that the function of each bmp gene has been conserved throughout evolution. As a step to characterize potential involvement of bmp genes in IB formation and development, spatiotemporal expressions of nine bmp genes (bmp2a, bmp2b, bmp3, bmp4, bmp5, bmp7b, bmp8a, bmp14 and bmp16) were investigated during the key development stages of IBs. During the ossification process from stage I (the IBs haven't emerged) to stage IV (all of the IBs ossified in the tail with the mature morphology), the expression profiles revealed that bmp16 was the most abundant transcript while bmp4 had the lowest abundance. The mRNA levels of bmp3, bmp4, bmp5 and bmp8a increased significantly at stage II, suggesting their roles in stimulating IB formation. The expression of bmp7b reached the highest level at stage III (the rapid period of IB development), suggesting potential involvement of bmp7b in promoting osteoblast differentiation. With the exception of bmp7b and bmp16, most bmp genes appeared a significant increase at IB maturation phase (stage IV), which means that they may play important roles in maintenance of IB morphogenesis. Spatial tissue distribution of bmp genes showed that most bmp genes were observed at the highest level in developing IBs at one year old fish. Spatiotemporal expression patterns suggest the potential key roles of these bmp genes in IBs formation and maintenance in fish, being as possible promoters or inhibitors.

Transcriptome comparison reveals insights into muscle response to hypoxia in blunt snout bream (Megalobrama amblycephala).

The economic and biological significance of blunt snout bream (Megalobrama amblycephala) makes this species important to explore the underlying molecular mechanism of hypoxia response. In the present study, we compared the transcriptional responses to serious hypoxia in skeletal muscle among hypoxia tolerant (MT), sensitive (MS) and control (without hypoxia treatment, MC) M. amblycephala obtained according to the time difference of losing balance after hypoxia treatment. A total of 88,200,889 clean reads were generated and assembled into 44,493 unigenes. Transcriptomic comparison revealed 463 genes differentially expressed among different groups. A similar hypoxia-induced transcription patterns suggested a common hypoxia response involved in cell cycle, p53 signaling pathway, apoptosis, heart contraction and blood circulation. Interesting, four genes, heat shock protein beta-8 (hspb8), cysteine/serine-rich nuclear protein 1 (csrnp1), salt-inducible kinase 1 (sik1), and visinin-like 1a (vsnl1a) were up-regulated in MT Vs MC but down-regulated in MS Vs MC. Additionally, FoxO signaling pathway was significantly enriched only in MT Vs MC. These results not only provided the first insights into the mechanism that muscle tissue coped with the hypoxia stress in cyprinid species, but offered a theory base for breeding of M. amblycephala with hypoxia-resistant traits.

Construction of a high-density linkage map and fine mapping of QTLs for growth and gonad related traits in blunt snout bream.

High-density genetic maps based on SNPs are essential for fine mapping loci controlling specific traits for fish species. Using restriction-site associated DNA tag sequencing (RAD-Seq) technology, we identified 42,784 SNPs evenly distributed across the Megalobrama amblycephala genome. Based on 2 parents and 187 intra-specific hybridization progenies, a total of 14,648 high-confidence SNPs were assigned to 24 consensus linkage groups (LGs) of maternal and paternal map. The total length of the integrated map was 3,258.38 cM with an average distance of 0.57 cM among 5676 effective loci, thereby representing the first high-density genetic map reported for M. amblycephala. A total of eight positive quantitative trait loci (QTLs) were detected in QTL analysis. Of that, five QTL explained ≥35% of phenotypic variation for growth traits and three QTL explained ≥16% phenotypic variation for gonad related traits. A total of 176 mapped markers had significant hits in the zebrafish genome and almost all of the 24 putative-chromosomes of M. amblycephala were in relatively conserved synteny with chromosomes of zebrafish. Almost all M. amblycephala and zebrafish chromosomes had a 1:1 correspondence except for putative-chromosome 4, which mapped to two chromosomes of zebrafish caused by the difference in chromosome numbers between two species.

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.

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.